Quinolone based compounds exhibiting, prolyl hydroxylase inhibitory activity, and compositions, and uses thereof

ABSTRACT

This invention relates to new quinolone based compounds that exhibit prolyl hydroxylase inhibitory activity. This invention also relates to methods of increasing HIF levels or activity in a subject or treating a condition associated with HIF levels or activity in a subject by administering to the subject at least one quinolone based compound. This invention further involves assays for the detection of a hydroxyproline residue in a HW molecule.

This application claims the benefit of priority of U.S. ProvisionalPatent Application No. 60/748,577, filed Dec. 9, 2005, and U.S.Provisional Patent Application No. 60/785,358, filed Mar. 24, 2006.

The cellular transcription factor HIF (Hypoxia Inducible Factor)occupies a central position in oxygen homeostasis in a wide range oforganisms and is a key regulator of responses to hypoxia. The genesregulated by HIF transcriptional activity can play critical roles inangiogenesis, erythropoiesis, hemoglobin F production, energymetabolism, inflammation, vasomotor function, apoptosis and cellularproliferation. HIF can also play a role in cancer, in which it iscommonly upregulated, and in the pathophysiological responses toischemia and hypoxia.

The HIF transcriptional complex comprises an αβ heterodimer: HIF-β is aconstitutive nuclear protein that dimerizes with oxygen-regulated HIF-αsubunits. Oxygen regulation occurs through hydroxylation of the HIF-αsubunits, which are then rapidly destroyed by the proteasome. Inoxygenated cells, the von Hippel-Lindau tumor suppressor protein (pVHL)binds to hydroxylated HIF-α subunits, thereby promoting their ubiquitindependent proteolysis. This process is suppressed under hypoxicconditions, stabilizing HIF-α and promoting transcriptional activationby the HIF αβ complex. See, e.g., U.S. Pat. No. 6,787,326.

Hydroxylation of HIF-α subunits can occur on proline and asparagineresidues and can be mediated by a family of 2-oxoglutarate dependentenzymes. This family includes the HIF prolyl hydroxylase isozymes(PHDs), which hydroxylate Pro 402 and Pro 564 of human HIF 1α, as wellas Factor Inhibiting HIF (FIH), which hydroxylates Asn 803 of human HIF1α. Inhibition of FIH or the PHDs leads to HIF stabilization andtranscriptional activation. See, e.g., Schofield and Ratcliffe, NatureRev. Mol. Cell Biol., Vol 5, pages 343-354 (2004).

Provided herein is at least one compound chosen from compounds ofFormula I:

a pharmaceutically acceptable salt thereof, a solvate thereof, a chelatethereof, a non-covalent complex thereof, a prodrug thereof, and mixturesof any of the foregoing, wherein:

n is 1 to 6;

R₁ is chosen from H, lower alkyl and substituted lower alkyl;

R₂ is chosen from H, lower alkyl and substituted lower alkyl;

R₃ and It_(t) are independently chosen from H, lower alkyl, substitutedlower alkyl, lower haloalkyl, substituted lower haloalkyl, or R₃ and R₄can join together to form a 3 to 6 membered ring or a substituted 3 to 6membered ring;

R₅ is chosen from OH, SH, NH₂, lower alkyl, substituted lower alkyl,lower alkoxy, substituted lower alkoxy, and sulfanyl;

R₆ is chosen from H, OH, SH, NH₂, NHSO₂R₁ and sulfonyl;

each of R₇, R₈, R₉ and R₁₀ is independently chosen from H, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, NR₃R₄, C(O)OH, OR₁₃, SR₁₃, SO₂R₁₃,CN, NO₂, halo, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heteroarylalkyl, substituted heteroarylalkyl,heterocycloalkyl, substituted heterocycloalkyl, alkylsilyl, substitutedalkylsilyl, alkynylsilyl, substituted alkynylsilyl, alkoxy, substitutedalkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, and —X—R₁₂, wherein:

R₃ and R₄ are defined above;

X is chosen from —N(R₁₁)—Y— and —Y—N(R₁₁)—;

Y is chosen from C(O), SO₂, alkylene, substituted alkylene, alkenylene,substituted alkenylene, alkynylene, and substituted alkynylene;

R₁₁ is chosen from H, lower alkyl, and substituted lower alkyl,

R₁₂ is chosen from H, heterocycloalkyl, substituted heterocycloalkyl,aryl, substituted aryl, heteroaryl, and substituted heteroaryl; and

R₁₃ is chosen from H, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl and NR₃R₄;

wherein at least one of adjacent pairs R₆ and R₇, R₇ and R₈, R₈ and R₉,R₉ and R₁₀, and R₁₀ and R₁, can join together to form a 4 to 7 memberedring or a substituted 4 to 7 membered ring.

Also provided herein is a pharmaceutical composition comprising at leastone pharmaceutically acceptable carrier, and a therapeutically effectiveamount of at least one compound described herein.

Further provided are pharmaceutical compositions comprising at least onepharmaceutically acceptable carrier, and a therapeutically effectiveamount of at least one compound described herein in combination with atleast one additional compound such as an erythropoiesis stimulatingagent or chemotherapeutic agent.

Additionally provided herein is a method of increasing HIF levels oractivity in a subject by administering to the subject at least onecompound described herein.

Further provided is a method of treating a condition where it is desiredto modulate HIF activity comprising administering to a subject at leastone compound described herein.

Also provided is a method of treating a hypoxic or ischemic relateddisorder in a subject comprising administering to a subject at least onecompound described herein.

Also provided is a method of treating anemia in a subject comprisingadministering to a subject at least one compound described herein.

Further provided is a method of modulating the amount of HIF in a cellcomprising contacting the cell with at least one compound describedherein.

Additionally provided is a method of increasing the amount of hemoglobinF in a subject comprising administering to the subject at least onecompound described herein.

Also provided is a method of modulating angiogenesis in a subjectcomprising administering to the subject at least one compound describedherein.

Additionally provided is a method of treating at least one disease in apatient in need of such treatment comprising administering to thepatient a therapeutically effective amount of at least one compounddescribed herein.

Also provided is a method of inhibiting HIF hydroxylation in a subjectcomprising administering to the subject at least one compound describedherein.

Further provided is an assay for the detection of HIF1α hydroxyprolineresidues comprising incubating a fluorochrome-labeled HIF1α polypeptideor fragment thereof with a VCB complex labeled with a rare earth elementand detecting the binding of the VCB complex to HIF1α by homogeneoustime-resolved FRET.

Also provided is an assay for the detection of HIF1α hydroxyprolineresidues comprising incubating a HIF1α polypeptide or fragment thereofwith a VCB complex labeled with ruthenium and detecting the binding ofthe VCB complex to HIF1α by electrochemiluminescence.

Additional embodiments of the invention are set forth in the descriptionwhich follows, or may be learned by practice of the invention.

FIG. 1 illustrates the ratio of fluorescence signal to backgroundgenerated by the interaction of Eu-VCB withstreptavidin-APC-hydroxyprolyl HIF1α peptide.

FIG. 2 illustrates the ratio of HTRF signal generated by the interactionof Eu-VCB with streptavidin-APC-hydroxyprolyl HIF1α peptide overbackground signal generated by the interaction of Eu-VCB withstreptavidin-APC-HIF1α peptide (nonhydroxylated). Panel A illustrates a0-125 nM peptide range. Panel B illustrates a 0-10 nM peptide range.

FIG. 3 illustrates VCB binding and HTRF detection for determining HIFPHD2 hydroxylation of a HIF1α peptide. Panel A illustrates a time coursefor the hydroxylation of the HIF1α peptide with increasing amounts ofHIF PHD2 enzyme. Panel B illustrates initial rates with increasingenzyme concentrations.

FIG. 4 illustrates the Ru-VCB/biotin-HIF—OH binding curve and linearrange determination by ECL detection.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the following specification andattached claims are approximations that may vary depending upon thestandard deviation found in their respective testing measurements.

As used herein, when any variable occurs more than one time in achemical formula, its definition on each occurrence is independent ofits definition at every other occurrence. When the chemical structureand chemical name conflict, the chemical structure is determinative ofthe identity of the compound. The compounds of the present disclosuremay contain one or more chiral centers and/or double bonds andtherefore, may exist as stereoisomers, such as double-bond isomers(i.e., geometric isomers), enantiomers or diastereomers. Accordingly,any chemical structures within the scope of the specification depicted,in whole or in part, with a relative configuration encompass allpossible enantiomers and stereoisomers of the illustrated compoundsincluding the stereoisomerically pure form (e.g., geometrically pure,enantiomerically pure or diastereomerically pure) and enantiomeric andstereoisomeric mixtures. Enantiomeric and stereoisomeric mixtures can beresolved into the component enantiomers or stereoisomers usingseparation techniques or chiral synthesis techniques well known to theskilled artisan.

Compounds of Formula I include, but are not limited to optical isomersof compounds of Formula I, racemates, and other mixtures thereof. Inthose situations, the single enantiomers or diastereomers, i.e.,optically active forms, can be obtained by asymmetric synthesis or byresolution of the racemates. Resolution of the racemates can beaccomplished, for example, by conventional methods such ascrystallization in the presence of a resolving agent, or chromatography,using, for example a chiral high-pressure liquid chromatography (HPLC)column. In addition, compounds of Formula I include Z- and E- forms (orcis- and trans-forms) of compounds with double bonds. Where compounds ofFormula I exists in various tautomeric forms, chemical entities of thepresent invention include all tautomeric forms of the compound.

Compounds of the present disclosure include, but are not limited tocompounds of Formula I and all pharmaceutically acceptable formsthereof. Pharmaceutically acceptable forms of the compounds recitedherein include pharmaceutically acceptable salts, solvates, crystalforms (including polymorphs and clathrates), chelates, non-covalentcomplexes, prodrugs, and mixtures thereof. In certain embodiments, thecompounds described herein are in the form of pharmaceuticallyacceptable salts. As used henceforth, the term “compound” encompassesnot only the compound itself, but also a pharmaceutically acceptablesalt thereof, a solvate thereof, a chelate thereof, a non-covalentcomplex thereof, a prodrug thereof, and mixtures of any of theforegoing.

As noted above, prodrugs also fall within the scope of chemicalentities, for example, ester or amide derivatives of the compounds ofFormula I. The term “prodrugs” includes any compounds that becomecompounds of Formula I when administered to a patient, e.g., uponmetabolic processing of the prodrug. Examples of prodrugs include, butare not limited to, acetate, formate, and benzoate and like derivativesof functional groups (such as alcohol or amine groups) in the compoundsof Formula I.

The term “solvate” refers to the compound formed by the interaction of asolvent and a compound. Suitable solvates are pharmaceuticallyacceptable solvates, such as hydrates, including monohydrates andhemi-hydrates.

“Alkenyl” refers to an unsaturated branched, straight-chain or cyclicalkyl group having at least one carbon-carbon double bond derived by theremoval of one hydrogen atom from a single carbon atom of a parentalkene. The group may be in either the Z- and E-forms (or cis or transconformation) about the double bond(s). Typical alkenyl groups include,but are not limited to, ethenyl; propenyls such as prop-1-en-1-yl,prop-1-en-2-yl, prop-2-en-l-yl (allyl), prop-2-en-2-yl,cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyls such asbut-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl; and thelike. In certain embodiments, an alkenyl group has from 2 to 20 carbonatoms and in other embodiments, from 2 to 6 carbon atoms, i.e. “loweralkenyl.”

“Alkynyl” refers to an unsaturated branched or straight-chain having atleast one carbon-carbon triple bond derived by the removal of onehydrogen atom from a single carbon atom of a parent alkyne. Typicalalkynyl groups include, but are not limited to, ethynyl; propynyl;butenyl, 2-pentynyl, 3-pentynyl, 2-hexynyl, 3-hexynyl and the like. Incertain embodiments, an alkynyl group has from 2 to 20 carbon atoms andin other embodiments, from 2 to 6 carbon atoms, i.e. “lower alkynyl.”

“Alkoxy” refers to a radical —OR where R represents an alkyl,substituted alkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, or substituted heteroaryl group as defined herein.Representative examples include, but are not limited to, methoxy,ethoxy, propoxy, butoxy, cyclohexyloxy, and the like.

“Alkoxycarbonyl” refers to a radical —C(O)—OR where R is as definedherein.

“Alkyl” refers to a saturated, branched or straight-chain monovalenthydrocarbon group derived by the removal of one hydrogen atom from asingle carbon atom of a parent alkane. Typical alkyl groups include, butare not limited to, methyl, ethyl, propyls such as propan-1-yl,propan-2-yl, and cyclopropan-1-yl, butyls such as butan-1-yl,butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,tert-butyl, and the like. In certain embodiments, an alkyl groupcomprises from 1 to 20 carbon atoms. As used herein the term “loweralkyl” refers to an alkyl group comprising from 1 to 6 carbon atoms.

“Aryl” refers to a monovalent aromatic hydrocarbon group derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Aryl encompasses 5- and 6-membered carbocyclicaromatic rings, for example, benzene; bicyclic ring systems wherein atleast one ring is carbocyclic and aromatic, for example, naphthalene,indane, and tetralin; and tricyclic ring systems wherein at least onering is carbocyclic and aromatic, for example, fluorene. For example,aryl includes 5- and 6-membered carbocyclic aromatic rings fused to a 5-to 7-membered heterocycloalkyl ring containing 1 or more heteroatomschosen from N, O, and S. In certain embodiments, an aryl group cancomprise from 6 to 10 carbon atoms. Aryl, however, does not encompass oroverlap in any way with heteroaryl, separately defined below. Hence, ifone or more carbocyclic aromatic rings is fused with a heterocycloalkylaromatic ring, the resulting ring system is heteroaryl, not aryl, asdefined herein.

“Arylalkyl” or “aralkyl” refers to an acyclic alkyl group in which oneof the hydrogen atoms bonded to a carbon atom, typically a terminal orsp³ carbon atom, is replaced with an aryl group. Typical arylalkylgroups include, but are not limited to, benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and thelike. Where specific alkyl moieties are intended, the nomenclaturearylalkyl, arylalkenyl, and/or arylalkynyl is used. In certainembodiments, an arylalkyl group can be (C₆₋₃₀) arylalkyl, e.g., thealkyl group of the arylalkyl group can be (C₁₋₁₀) and the aryl moietycan be (C₅₋₂₀).

“Carbonyl” refers to a radical —C(O) group.

“Carboxy” refers to the radical —C(O)OH.

“Cyano” refers to the radical —CN.

“Cycloalkyl” refers to a saturated or unsaturated cyclic alkyl group.Where a specific level of saturation is intended, the nomenclature“cycloalkanyl” or “cycloalkenyl” is used. Typical cycloalkyl groupsinclude, but are not limited to, groups derived from cyclopropane,cyclobutane, cyclopentane, cyclohexane, and the like. In certainembodiments, the cycloalkyl group can be C₃₋₁₀ cycloalkyl, such as, forexample, C₃₋₆ cycloalkyl.

“Heterocycloalkyl” refers to a saturated or unsaturated, butnon-aromatic, cyclic alkyl group in which one or more carbon atoms (andany associated hydrogen atoms) are independently replaced with the sameor different heteroatom and its associated hydrogen atoms, whereappropriate. Typical heteroatoms to replace the carbon atom(s) include,but are not limited to, N, P, O, S, and Si. Where a specific level ofsaturation is intended, the nomenclature “heterocycloalkanyl” or“heterocycloalkenyl” is used. Typical heterocycloalkyl groups include,but are not limited to, groups derived from epoxides, imidazolidine,morpholine, piperazine, piperidine, pyrazolidine, pyrrolidine,quinuclidine, tetrahydrofuran, tetrahydropyran and the like. Substitutedheterocycloalkyl also includes ring systems substituted with one or moreoxo (═O) or oxide (—O⁻) substituents, such as piperidinyl N-oxide,morpholinyl-N-oxide, 1-oxo-1-thiomorpholinyl and1,1-dioxo-1-thiomorpholinyl.

“Disease” refers to any disease, disorder, condition, symptom, orindication.

“Halo” refers to a fluoro, chloro, bromo, or iodo group.

“Heteroaryl” refers to a monovalent heteroaromatic group derived by theremoval of one hydrogen atom from a single atom of a parentheteroaromatic ring system. Heteroaryl encompasses:

-   -   5- to 7-membered aromatic, monocyclic rings containing one or        more, for example, from 1 to 4, or in certain embodiments, from        1 to 3, heteroatoms chosen from N, O, and S, with the remaining        ring atoms being carbon; and    -   polycyclic heterocycloalkyl rings containing one or more, for        example, from 1 to 4, or in certain embodiments, from 1 to 3,        heteroatoms chosen from N, O, and S, with the remaining ring        atoms being carbon and wherein at least one heteroatom is        present in an aromatic ring.

For example, heteroaryl includes a 5- to 7-membered heteroaromatic ringfused to a 5- to 7-membered cycloalkyl ring and a 5- to 7-memberedheteroaromatic ring fused to a 5- to 7-membered heterocycloalkyl ring.For such fused, bicyclic heteroaryl ring systems wherein only one of therings contains one or more heteroatoms, the point of attachment may beat the heteroaromatic ring or the cycloalkyl ring. When the total numberof S and O atoms in the heteroaryl group exceeds 1, those heteroatomsare not adjacent to one another. In certain embodiments, the totalnumber of S and O atoms in the heteroaryl group is not more than 2. Incertain embodiments, the total number of S and O atoms in the aromaticheterocycle is not more than 1. Heteroaryl does not encompass or overlapwith aryl as defined above. Typical heteroaryl groups include, but arenot limited to, groups derived from acridine, arsindole, carbazole,β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole,indole, indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene, and the like. In certain embodiments, theheteroaryl group can be between 5 to 20 membered heteroaryl, such as,for example, a 5 to 10 membered heteroaryl. In certain embodiments,heteroaryl groups can be those derived from thiophene, pyrrole,benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole,oxazole, and pyrazine.

“Heteroarylalkyl” or “heteroaralkyl” refers to an acyclic alkyl group inwhich one of the hydrogen atoms bonded to a carbon atom, typically aterminal or sp³ carbon atom, is replaced with a heteroaryl group. Wherespecific alkyl moieties are intended, the nomenclatureheteroarylalkanyl, heteroarylalkenyl, and/or heteroarylalkynyl is used.In certain embodiments, the heteroarylalkyl group can be a 6 to 30membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moietyof the heteroarylalkyl can be 1 to 10 membered and the heteroaryl moietycan be a 5 to 20-membered heteroaryl.

“Sulfonyl” refers to a radical —S(O)₂R where R is an alkyl, substitutedalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substitutedaryl, or substituted heteroaryl group as defined herein. Representativeexamples include, but are not limited to methylsulfonyl, ethylsulfonyl,propylsulfonyl, butylsulfonyl, and the like.

“Sulfanyl” refers to a radical —SR where R is an alkyl, substitutedalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substitutedaryl, or substituted heteroaryl group as defined herein that may beoptionally substituted as defined herein. Representative examplesinclude, but are not limited to, methylthio, ethylthio, propylthio,butylthio, and the like.

“Pharmaceutically acceptable” refers to generally recognized for use inanimals, and more particularly in humans.

“Pharmaceutically acceptable salt” refers to a salt of a compound thatis pharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. Such salts include: (1)acid addition salts, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, andthe like; or formed with organic acids such as acetic acid, propionicacid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvicacid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, and the like; or (2) salts formed when an acidicproton present in the parent compound either is replaced by a metal ion,e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; orcoordinates with an organic base such as ethanolamine, diethanolamine,triethanolamine, N-methylglucamine, dicyclohexylamine, and the like.

“Pharmaceutically acceptable excipient,” “pharmaceutically acceptablecarrier,” or “pharmaceutically acceptable adjuvant” refer, respectively,to an excipient, carrier or adjuvant with which at least one compound ofthe present disclosure is administered. “Pharmaceutically acceptablevehicle” refers to any of a diluent, adjuvant, excipient or carrier withwhich at least one compound of the present disclosure is administered.

“Stereoisomer” refers to an isomer that differs in the arrangement ofthe constituent atoms in space. Stereoisomers that are mirror images ofeach other and optically active are termed “enantiomers,” andstereoisomers that are not mirror images of one another and areoptically active are termed “diastereoisomers.”

“Subject” includes mammals and humans. The terms “human” and “subject”are used interchangeably herein.

“Substituted” refers to a group in which one or more hydrogen atoms areeach independently replaced with the same or different substituent(s).Typical substituents include, but are not limited to, —X, —R₃₃, —OH, ═O,OR₃₃, —SR₃₃, —SH, ═S, —NR₃₃R₃₄, ═NR₃₃, —CX₃, —CF₃, —CN,—NO₂,—S(O)₂R₃₃,—OS(O₂)OH, —OS(O)₂R₃₃, —OP(O)(OR₃₃)(OR₃₄), —C(O)R₃₃, —C(S)R₃₃,—C(O)OR₃₃, —C(O)NR₃₃R₃₄, —C(O)OH, —C(S)OR₃₃, —NR₃₅C(O)NR₃₃R₃₄,—NR₃₅C(S)NR₃₃R₃₄, —NR₃₅C(NR₃₃)NR₃₃R₃₄, —C(NR₃₃)NR₃₃R₃₄, —S(O)₂NR₃₃R₃₄,—NR₃₅S(O)₂R₃₃, —NR₃₅C(O)R₃₃, and —S(O)R₃₃ where each X is independentlya halo; each R₃₃ and R₃₄ are independently hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substitutedheterocycloalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl,substituted heteroarylalkyl, —NR₃₅R₃₆, —C(O)R₃₅ or —S(O)₂R₃₅ oroptionally R₃₃ and R₃₄ together with the atom to which R₃₃ and R₃₄ areattached form one or more heterocycloalkyl, substitutedheterocycloalkyl, heteroaryl, or substituted heteroaryl rings; and R₃₅and R₃₆ are independently hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl,substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl,heteroaryl, substituted heteroaryl, heteroarylalkyl or substitutedheteroarylalkyl, or optionally R₃₅ and R₃₆ together with the nitrogenatom to which R₃₅ and R₃₆ are attached form one or moreheterocycloalkyl, substituted heterocycloalkyl, heteroaryl, orsubstituted heteroaryl rings. In certain embodiments, a tertiary amineor aromatic nitrogen may be substituted with on or more oxygen atoms toform the corresponding nitrogen oxide.

“Therapeutically effective amount” refers to the amount of a compoundthat, when administered to a subject for treating a disease, or at leastone of the clinical symptoms of a disease or disorder, is sufficient toaffect such treatment for the disease, disorder, or symptom. The“therapeutically effective amount” can vary depending on the compound,the disease, disorder, and/or symptoms of the disease or disorder,severity of the disease, disorder, and/or symptoms of the disease ordisorder, the age of the subject to be treated, and/or the weight of thesubject to be treated. An appropriate amount in any given instance canbe readily apparent to those skilled in the art or capable ofdetermination by routine experimentation.

“Treating” or “treatment” of any disease or disorder refers to arrestingor ameliorating a disease, disorder, or at least one of the clinicalsymptoms of a disease or disorder, reducing the risk of acquiring adisease, disorder, or at least one of the clinical symptoms of a diseaseor disorder, reducing the development of a disease, disorder or at leastone of the clinical symptoms of the disease or disorder, or reducing therisk of developing a disease or disorder or at least one of the clinicalsymptoms of a disease or disorder. “Treating” or “treatment” also refersto inhibiting the disease or disorder, either physically, (e.g.,stabilization of a discernible symptom), physiologically, (e.g.,stabilization of a physical parameter), or both, or inhibiting at leastone physical parameter which may not be discernible to the subject.Further, “treating” or “treatment” refers to delaying the onset of thedisease or disorder or at least symptoms thereof in a subject which maybe exposed to or predisposed to a disease or disorder even though thatsubject does not yet experience or display symptoms of the disease ordisorder.

Reference will now be made in detail to embodiments of the presentdisclosure. While certain embodiments of the present disclosure will bedescribed, it will be understood that it is not intended to limit theembodiments of the present disclosure to those described embodiments. Tothe contrary, reference to embodiments of the present disclosure isintended to cover alternatives, modifications, and equivalents as may beincluded within the spirit and scope of the embodiments of the presentdisclosure as defined by the appended claims.

Embodiments of the present invention are directed to at least onecompound of Formula I:

a pharmaceutically acceptable salt thereof, a solvate thereof, a chelatethereof, a non-covalent complex thereof, a prodrug thereof, and mixturesof any of the foregoing, wherein:

n is 1 to 6;

R₁ is chosen from H, lower alkyl and substituted lower alkyl;

R₂ is chosen from H, lower alkyl and substituted lower alkyl;

R₃ and R₄ are independently chosen from H, lower alkyl, substitutedlower alkyl, lower haloalkyl, substituted lower haloalkyl, or R₃ and R₄can join together to form a 3 to 6 membered ring or a substituted 3 to 6membered ring;

R₅ is chosen from OH, SH, NH₂, lower alkyl, substituted lower alkyl,lower alkoxy, substituted lower alkoxy, and sulfanyl;

R₆ is chosen from H, OH, SH, NH₂, NHSO₂R₁ and sulfonyl;

each of R₇, R₈, R₉ and R₁₀ is independently chosen from H, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, NR₃R₄, C(O)OH, OR₁₃, SR₁₃, SO₂R₁₃,CN, NO₂, halo, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heteroarylalkyl, substituted heteroarylalkyl,heterocycloalkyl, substituted heterocycloalkyl, alkylsilyl, substitutedalkylsilyl, alkynylsilyl, substituted alkynylsilyl, alkoxy, substitutedalkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, and —X—R₁₂, wherein:

R₃ and R₄ are defined above;

X is chosen from —N(R₁₁)—Y— and —Y—N(R₁₁)—;

Y is chosen from C(O), SO₂, alkylene, substituted alkylene, alkenylene,substituted alkenylene, alkynylene, and substituted alkynylene;

R₁₁ is chosen from H, lower alkyl, and substituted lower alkyl,

R₁₂ is chosen from H, heterocycloalkyl, substituted heterocycloalkyl,aryl, substituted aryl, heteroaryl, and substituted heteroaryl; and

R₁₃ is chosen from H, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl and NR₃R₄;

wherein at least one of adjacent pairs R₆ and R₇, R₇ and R_(g), R₈ andR₉, R₉ and R₁₀, and R₁₀ and R₁, can join together to form a 4 to 7membered ring or a substituted 4 to 7 membered ring.

In certain embodiments of compounds of Formula I, R₁ is chosen from alower alkyl such as methyl or ethyl.

In certain embodiments of compounds of Formula I, R₂ is chosen from H.

In certain embodiments of compounds of Formula I, R₃ and R₄ areindependently chosen from H, lower alkyl such as methyl or ethyl,substituted lower alkyl and substituted hydroxyalkyl such ashydroxymethyl.

In certain embodiments of compounds of Formula I, R₅ is chosen from OH,a lower alkoxy such as methoxy, ethoxy and propoxy, a substituted loweralkoxy and a primary amide.

In certain embodiments of compounds of Formula I, R₆ is chosen from H,OH and alkoxy.

In certain embodiments of compounds of Formula I, R₃ and R₄ jointogether to form a 3 to 6 membered ring or a substituted 3 to 6 memberedring. The 3 to 6 membered rings can comprise at least one heteroatom,such as at least two heteroatoms.

In certain embodiments of compounds of Formula I, R₆ and R₇ can jointogether to form a 4 to 7 membered ring or a substituted 4 to 7 memberedring. The 4 to 7 membered rings can comprise at least one heteroatom,such as at least two heteroatoms, and at least three heteroatoms.

In certain embodiments of compounds of Formula I, at least one of R₇,R₈, R₉ and R₁₀ is independently chosen from halo and a moietysubstituted with at least one halo, such as trifluoromethyl.

In certain embodiments of compounds of Formula I, at least one of R₇,R₈, R₉ and R₁₀ is independently chosen from alkoxy or substitutedalkoxy.

In certain embodiments of compounds of Formula I, at least one of R₇,R₈, R₉ and R₁₀ is independently chosen from alkylsilyl, substitutedalkylsilyl, alkynylsilyl, and substituted alkynylsilyl.

In certain embodiments of compounds of Formula I, at least one of R₇,R₈, R₉ and R₁₀ is independently chosen from aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocycloalkyl, and substitutedheterocycloalkyl, such as substituted pyridines, substitutedpyrimidines, substituted pyrazines, substituted pyridazines, substitutedtetrahydrofurans and substituted piperidines

In certain embodiments of compounds of Formula I, at least one of R₇,R₈, R₉ and R₁₀ is independently chosen from H, alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl, such asisopropyl, cyclohexane, cyclopentane, cyclohexene and cyclopentene.

Examples of individual representative compounds of the presentdisclosure, and compounds comprised in compositions of the presentdisclosure, and used in methods of the present disclosure are listed inTable 1. Each compound listed in Table 1, i.e., Compounds 1-175,contains information directed to its structure, name, molecular weight,hydrogen NMR data and at least one method of synthesis.

In certain embodiments, compounds of the present disclosure inhibitprolyl hydroxylases such as HIF prolyl hydroxylases. The assays of thepresent disclosure may be used to determine the prolyl hydroxylaseinhibitory activity of a compound.

In certain embodiments, compounds of the present disclosure modulate HIFlevels or activity, for example, by stabilizing HIF.

Furthermore, compounds of the present disclosure can contain one or morechiral centers. Such compounds can be prepared or isolated as purestereoisomers, i.e., as individual enantiomers or diastereomers, or asstereoisomer-enriched mixtures. All such stereoisomers, and enrichedmixtures thereof, are included within the scope of the presentdisclosure. Pure stereoisomers, and enriched mixtures thereof, can beprepared using, for example, optically active starting materials orstereoselective reagents well-known in the art. Alternatively, racemicmixtures of such compounds can be separated using, for example, chiralcolumn chromatography, chiral resolving agents and the like.

Certain embodiments of the present disclosure are directed to apharmaceutical composition comprising at least one pharmaceuticallyacceptable excipient, and a therapeutically effective amount of at leastone compound described herein. The at least one compound can be presentin an amount effective for the treatment of at least one disease chosenfrom ischemia, anemia, wound healing, auto-transplantation,allo-transplantation, xeno-transplantation, systemic high bloodpressure, thalassemia, diabetes, cancer and an inflammatory disorder.

Other embodiments of the present disclosure are directed to a method oftreating a condition where it is desired to modulate HIF activitycomprising administering to a subject at least one compound describedherein. The condition can be chosen from at least one of ischemia,anemia, wound healing, auto-transplantation, allo-transplantation,xeno-transplantation, systemic high blood pressure, thalassemia,diabetes, cancer and an inflammatory disorder.

A further embodiment is directed to a method of treating at least onedisease in a patient in need of such treatment comprising administeringto the patient a therapeutically effective amount of at least onecompound described herein. The at least one disease can be chosen fromischemia, anemia, wound healing, auto-transplantation,allo-transplantation, xeno-transplantation, systemic high bloodpressure, thalassemia, diabetes, cancer and an inflammatory disorder.

Other embodiments of the present disclosure are directed to assays forthe detection of hydroxyprolyl HIF1α proteins or fragments thereofcomprising incubating a fluorochrome-labeled HIF1α polypeptide orfragment thereof with a VCB complex labeled with a rare earth elementand detecting the binding of the VCB complex to HIF1α by homogeneoustime-resolved FRET. In certain embodiments, the fluorochrome may beallophycocyanin. In other embodiments, the rare earth element may beeuropium.

Additional embodiments are directed to assays for the detection ofhydroxyprolyl HIF1α proteins or fragments thereof comprising incubatinga HIF1α polypeptide or fragment thereof with a VCB complex labeled withruthenium and detecting the binding of the VCB complex to HIF1α byelectrochemiluminescence. In certain embodiments, the -HIF1α polypeptideor fragment thereof may be bound to a solid support.

The assays of the present disclosure may also be used to detect thehydroxylation of HIF1α proteins or fragments thereof by HIF prolylhydroxylases.

Further embodiments of the present disclosure are directed to assays forinhibitors of HIF prolyl hydroxylases.

The compounds of the present invention can be produced by one or more ofthe following general reaction schemes.

The following are examples of methods that can be used to produceintermediates to and compounds of the present invention.

Method 1: 5-Iodo-2-(methylamino)benzoic acid

In a 1L 3-neck flask was added 2-(methylamino)benzoic acid (40 g, 265mmol), water (300 ml), and Hydrochloric acid (26.7 ml, 871 mmol). Asolution of iodine monochloride was prepared by adding iodinemonochloride (43 g, 265 mmol) to a cooled solution (0° C.) ofHydrochloric acid (45 ml, 1469 mmol) and water (167 ml, 9272 mmol). Theiodine monochloride solution was added rapidly to the stirred solutionof the 2-(methylamino)benzoic acid. The mixture was allowed to stir for2 hrs, filtered on a medium frit funnel and the solids washed with waterand dried under vacuum to give a quantitative yield of the product as alight-green powder. Ref. McDowell, R. S. et al, JACS, 1994, 116,5077-5083.

Method 2: 6-Iodo-1-methyl-1H-benzo[d][1,3]oxazine-2,4-dione

To a stirred solution of 5-iodo-2-(methylamino)benzoic acid (10 g, 36mmol), sodium carbonate (4 g, 36 mmol) and water (130 ml, 7218 mmol),cooled to 0° C., was slowly added, via addition funnel, a 2M phosgene(18 ml, 36 mmol) solution in toluene. After 2 hrs, the precipitatedproduct was isolated by filtration. The solids were washed with 100 mlof water, 150 ml of a 1:1 mixture ethanol and ether, 100 ml of ether,and dried under vacuum to give the desired product. Yield=7.15 g.

Method 3: 5-chloro-1H-benzo [d][1,3]oxazine-2,4-dione

In a 250 mL round-bottom flask under N₂ was dissolved2-amino-6-chlorobenzoic acid (11.69 g, 68 mmol) in 100 mL of1,4-dioxane. The solution was cooled to 0° C. and to this solution wasadded phosgene (36 ml, 68 mmol) via a dropping funnel. The reactionmixture was stirred for 24 hours allowing to warm to 23° C. (rt). Theresulting white solid was filtered off and washed with 1,4-dioxane andEt₂O. Yield=12.5g, 93%

Method 4: 6-Bromo-1-methyl-1H-benzo[d][1,3]oxazine-2,4-dione

5-bromoisatoic anhydride (3 g, 12 mmol) was stirred in 50 mL of DMF at0° C. and sodium hydride (60% dispersion in mineral oil) (0.4 g, 15mmol) was added in portions, with stirring for 1 hour at roomtemperature. lodomethane (0.8 mL, 12 mmol) was added drop wise and thereaction mixture was allowed to stir for 4 hours. Water (50 mL) wasadded slowly and 50 mL of Dichloromethane (DCM) was also added. A whitesolid precipitated out and was filtered off. The layers were separatedlayers. Ageous layer extracted with DCM (2×25ml). The combined organiclayers were extracted with water (4×25 ml) and once with brine (25 ml).

The organic layer was dried with MgSO₄ and the solvent removed. Theresidue was purified by flash chromatography (0-3% MeOH/DCM) to afford1.57 g of product. Yield 49%

Method 5: 7-Bromo-1-methyl-1H-benzo[d][1,3]oxazine-2,4-dione

Sodium hydride (0.47 g, 12 mmol) was added to a 3 neck 250 mL RBF undernitrogen and then washed with hexanes. Once the hexanes were decanted,N,N-dimethylformamide (20.0 mL, 11 mmol) was added. The resultingmixture was cooled to 0° C. using an ice-water bath, and then7-bromo-1H-benzo[d][1,3]oxazine-2,4-dione (2.7 g, 11 mmol) was added inone batch. After stirring at room temperature for 1 hour, iodomethane(0.70 mL, 11 mmol) was added dropwise to the yellow solution, and thereaction mixture was stirred for 16 hours. Water (50 mL) was added, andthe resulting precipitate that formed was collected via filtration. Thesolid was washed with additional water (100 mL), followed by ether (100mL). Drying in a vacuum oven overnight at 50° C. afforded the desiredproduct as an off-white solid (2.1 g, 74% yield).

Method 6: methyl1-methyl-2,4-dioxo-2,4-dihydro-1H-benzo[d][1,3]oxazine-7-carboxylate

Sodium hydride (0.51 g, 21 mmol) was added to chilled (0° C.) DMF (40ml). The 2,4-dioxo-2,4-dihydro-1H-benzo[d][1,3]oxazine-7-carboxylic acid(2.0 g, 9.7 mmol) was added to this mixture and stirred at 0° C. untilhydrogen gas evolution (vigorous) ceased. A yellow suspension resulted.To this mixture, iodomethane (1.2 ml, 19 mmol) was then added and themixture was warmed to room temperature, followed by heating to 50° C.for 30 min. The mixture was cooled to 0° C. and water was added slowlyfollowed by dichloromethane. The layers were separated and the aqueouslayer was extracted with dichloromethane 3×. The combined organic layerswere washed sat. NaHCO₃ (10 ml) 2× with H₂O, and sat. NaCl (15 ml). Theorganic layer was dried over MgSO4, filtered and concentrated to give ayellow solution in DMF which was used without purification.

Method 7: Ethyl4-hydroxy-6-iodo-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylate

60% sodium hydride (1.2 ml, 28 mmol) was added portionwise to a mixtureof diethyl ester malonic acid (17 ml, 110 mmol) andN,N-Dimethylformamide (75 ml) with stirring at room temperature. Amixture of 6-iodo-1-methyl-1H-benzo[d][1,3]oxazine-2,4-dione (7.12 g, 23mmol) and N,N-Dimethylformamide (75 ml) was added to this solutionfollowed by stirring at 120° C. for 2.5 hours. The precipitate thatformed was collected by filtration and dissolved in water and 30% HClwas added to the mixture. The precipitated crystals were collected byfiltration and dried to give the desired product. Yield=3.3 g.

Method 8: tert-Butyl4-hydroxy-6-iodo-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylate

To a solution of tert-butyl malonate (5 ml, 20 mmol) in 1,4-Dioxane (70ml) was added 60% Sodium hydride (0.8 g, 35 mmol) in portions. Themixture was stirred at room temperature for 45 min. then a solution of6-iodo-1-methyl-1H-benzo[d][1,3]oxazine-2,4-dione (6.1 g, 20 mmol) in1,4-Dioxane (40 ml) was added. The mixture was placed in an oil bath at60° C. and the bath temp was raised to 120° C. over a period of 20 minafter which stirring was continued for 90 min. The solvent was removedon a roto-evaporator and cold water (300 ml) was added to the residue.The mixture was washed with DCM (100 ml) then the aqueous phase wasacidified with 2N HCl. The organic layer was extracted into DCM (2×100ml) and after drying over MgSO4 the solvent was removed on aroto-evaporator. Yield=4.1 g.

Method 9: Methyl7-bromo-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylate

To a 50 mL RBF was added sodium hydride (0.15 g, 3.7 mmol) andN,N-dimethylformamide (50 mL, 3.1 mmol) under nitrogen. The mixture wascooled with an ice-water bath for 10 min, and then dimethyl malonate(6.4 mL, 56 mmol) was added over 3 min. A mixture of7-bromo-1-methyl-1H-benzo[d][1,3]oxazine-2,4-dione (0.80 g, 3.1 mmol) inDMF (5.0 mL) was added, and then the reaction was placed in oil bath at120° C. for 3 hours. The reaction was cooled to room temperature, andwater (25 mL) was added to the mixture. A white solid was collected byfiltration and washed with water (100 mL), followed by ether (100 mL).The white solid was placed in vacuum oven at 50° C. for 6 h to affordthe desired product as a white solid (0.65 g, 67%).

Method 10: 3-benzyl 7-methyl4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3,7-dicarboxylate

To a solution of dibenzyl malonate (2.99 ml, 11.9 mmol) in DMF (41 ml)was added sodium hydride in portions. The cloudy grey mixture wasstirred at room temperature for 20 min after which time a clear solutionresulted. The solution was further stirred at 120° C. for 20 min beforeadding a solution of methyl1-methyl-2,4-dioxo-2,4-dihydro-1H-benzo[d][1,3]oxazine-7-carboxylate(2.82 g, 11.9 mmol) in DMF (41 ml). The resulting yellow solution wasstirred at 120° C. for 3 hrs. The reaction mixture was cooled to roomtemperature, 2N HCl and EtOAc were added and the layers were separated.The aqueous layer was extracted with EtOAc 3×, the combined organicswere washed with H₂O (1×) followed by sat. NaCl (2×). The organic phasewas then dried over MgSO₄, filtered and concentrated to give a yellowsolid. Purification was performed by ISCO using 10% to 50% Hex/EtOAcgradient, 40 g column to give 300 mg of a yellow solid.

Method 11:4-Hydroxy-6-iodo-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid

To a solution of tert-butyl4-hydroxy-6-iodo-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylate (4.1g, 10 mmol) and Acetonitrile (20 ml), cooled in an ice bath, was added70% perchloric acid (0.2 ml) and the mixture was stirred 30 sec. Ayellow solid was filtered. Yield=0.5 g.

Method 12:4-hydroxy-7-(methoxycarbonyl)-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylicacid

Palladium, 10 wt. % on activated carbon, (5.1 mg, 48 μmol) was added toa solution of 3-benzyl 7-methyl4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3,7-dicarboxylate (88 mg,240 μmol) in ethyl acetate (19 ml) and brought under an atmosphere of H₂while stirring vigorously. After the reaction was complete, the mixturewas filtered through a Celite pad, rinsing with EtOAc/DCM to give anoff-white powder (59 mg, 89%) which was used without furtherpurification.

Method 13: methyl3-((2-(benzyloxy)-2-oxoethyl)carbamoyl)-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-7-carboxylate

4-hydroxy-7-(methoxycarbonyl)-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylicacid (107 mg, 386 μmol), glycine benzyl ester hydrochloride (117 mg, 579μmol), pybop (603 mg, 1158 μmol), and diisopropylethylamine (403 μl,2316 μmol) were dissolved in DMF and stirred at room temperature for 24hours. The reaction mixture was diluted with H₂O and DCM, the layerswere separated and the aqueous layer was extracted with DCM (3×), theorganics were washed with H₂O (2×) and dried over MgSO₄, filtered andconcentrated to yield a yellow solid. Flash column chromatography wasperformed using 2:1 Hex/EtOAc to give 45 mg of desired product.

Method 14: Ethyl2-(4-hydroxy-6-iodo-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate(glycine methyl ester or glycine t-butyl ester can also be used)

A solution of ethyl4-hydroxy-6-iodo-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylate (0.5g, 1 mmol), glycine ethyl ester hydrochloride (0.2 g, 1 mmol) and1,4-Dioxane (30 ml) in a 100 ml round-bottom flask, equipped with ashort-path distillation head, was heated to 120° C. After 7 hrs,reaction was complete. The solvent was completely distilled over. A tansolid residue was washed with EtOAc and concentrated on aroto-evaporator, then on high vacuum, for 15 hrs. A tan solid was washedwith DCM and a white solid was filtered off. The filtrate wasconcentrated on a roto-evaporator to give a light tan solid. Yield=0.4g.

Method 15: Methyl2-(4-hydroxy-6-iodo-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate(glycine methyl ester or glycine t-butyl ester can also be used)

To a 100 ml rb flask was added4-hydroxy-6-iodo-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid(0.5 g, 1 mmol), glycine methyl ester hydrochloride (0.3 g, 2 mmol),Pybop (1 g, 2 mmol), N,N-Dimethylformamide (10 ml) and Triethylamine(0.6 ml, 4 mmol), and the mixture was stirred at room temperature. After4 hrs, additional glycine methyl ester hydrochloride (0.3 g, 2 mmol) andTriethylamine (0.6 ml, 4 mmol) were added. After 1 hour, more Pybop (1g, 2 mmol) was added. The mixture was stirred for 3 days, and then awhite solid was filtered. Yield=0.28 g.

Method 16: (S)-methyl2-(4-hydroxy-8-methoxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)propanoate

In a 35 mL sealed vial under N₂ was suspended methyl4-hydroxy-8-methoxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylate(0.50 g, 1.9 mmol) in 1,4-dioxane (15 mL). To this solution was addedL-alanine methyl ester hydrochloride (0.29 g, 2.1 mmol) and the reactionmixture was stirred at 120° C. overnight (18 h). The solution wasremoved from the heat and filtered over a fine frit funnel to remove anyundissolved starting material. The filtrate was concentrated in vacuoand the remaining precipitate was suspended in Et₂O, filtered and washedwith Et₂O and dried to provide a light yellow solid. Yield=0.40 g, 63%.

Method 17: Methyl2-(1-ethyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate

In 4 mL of toluene in a microwave vial, ethyl1-ethyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylate (0.380 g, 1mmol), glycine methyl ester hydrochloride (0.5 g, 4 mmol) was microwavedat 180° C. for 3 minutes and then purified by silica flashchromatography (with a 1-5% MeOH/DCM gradient to afford 0.050 g. Yield:11%

Method 18: Ethyl2-(4-hydroxy-1-methyl-2-oxo-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2-dihydroquinoline-3-carboxamido)acetate

A mixture of ethyl2-(4-hydroxy-6-iodo-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate(5 g, 12 mmol), bis(pinacolato)diboron (3 g, 13 mmol),1,1′-bis(diphenylphosphino)ferrocene-palladium dichloride (0.3 g, 0.5mmol), acetic acid, potassium salt (1 ml, 23 mmol), and 1,4-Dioxane (100ml) was heated to 85-95° C. under an atmosphere of nitrogen. After 44hrs, cooled reaction mixture and filtered off purplish-beige solid. Thefiltrate was concentrated on a roto-evaporator and treated with EtOH. Atan solid was filtered off and washed with ether. A second crop wasobtained from the filtrate mixture. Yield=2.5 g.

Method 19: Ethyl2-(4-hydroxy-1-methyl-2-oxo-6-phenyl-1,2-dihydroquinoline-3-carboxamido)acetate

A solution of ethyl2-(4-hydroxy-6-iodo-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate(200 mg, 465 μmol), phenylboronic acid (85 mg, 697 μmol), 2M Sodiumcarbonate (0.7 ml, 1395 μmol), Tetrakis(triphenylphosphine) palladium(0)(5 mg, 5 μmol) and N,N-Dimethylformamide (10 ml) was stirred at 100° C.After 8 hrs, an additional equivalent of the phenyl boronic acid wasadded, and the mixture was stirred for 15 hrs. The reaction mixture wasconcentrated on a roto-evaporator and extracted with EtOAc. This productwas then washed with water and brine, then dried with MgSO₄ andconcentrated on a roto-evaporator to give the crude product as ared-orange oil. The crude product was purified by silica flashchromatography (10-75% EtOAc:Hex step gradient) to give the desiredproduct as a white solid. Yield=120 mg. In some cases, ester hydrolysiswas observed and the carboxylic acid was isolated.

Method 20: Ethyl2-(6-(6-chloropyrimidin-4-yl)-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate

To a 10 ml reaction vial was charged ethyl2-(4-hydroxy-1-methyl-2-oxo-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2-dihydroquinoline-3-carboxamido)acetate(200 mg, 465 μmol) in 1,4-dioxane (5 ml), 4,6-dichloropyrimidine (69 mg,465 μmol), tetrakis(triphenylphosphine) palladium(0) (27 mg, 23 μmol)and sodium carbonate (0.7 ml, 1395 μmol), and the reaction vial washeated to 70° C. After reaction was complete, the reaction mixture wasconcentrated on a roto-evaporator, extracted with EtOAc, washed withwater and brine (3× ea.) then dried with MgSO₄ and concentrated on aroto-evaporator. The yellow solid was washed with EtOH and filtered andthe solid was washed with ether. Yield=55 mg.

Method 21: Methyl2-(7-(3,5-dimethylisoxazol-4-yl)-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate

A mixture of methyl2-(7-bromo-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate(400 mg, 1084 μmol), 3,5-dimethylisoxazol-4-ylboronic acid (305 mg, 2167μmol) and Pd(PPh₃)₄ (125 mg, 108 μmol) in 8 ml 1,2-dimethoxyethane (orDMF) and 1.6 ml 2M aqueous Na₂CO₃ was heated to 75° C. and stirred for12 hours. The mixture was cooled to 24° C., treated with 1M aqueous HCland CHCl₃, after which solids precipitated. The organic layer wasseparated and the solids were collected by filtration, and washed withMeOH.

Method 22:2-(4-Hydroxy-1-methyl-2-oxo-6-(piperidin-1-yl)-1,2-dihydroquinoline-3-carboxamido)aceticacid

A solution of ethyl2-(4-hydroxy-6-iodo-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate(250 mg, 581 μmol), Pd₂dba₃.CHCl₃ (60 mg, 58 μmol), X-Phos (55 mg, 116μmol) and sodium t-butoxide (279 mg, 2906 μmol) in 1,4-dioxane (5 ml)was treated with piperidine (287 μl, 2906 μmol). The reaction wasstirred at 80° C. in a sealed tube. After 22 hours, the solution wascooled to 23° C., filtered through celite (washing with MeOH),concentrated, diluted with methanol/DMSO and purified by RP HPLC (0-100%MeCN/water+1% TFA, 10 min), affording 17 mg (8%) of2-(4-hydroxy-1-methyl-2-oxo-6-(piperidin-1-yl)-1,2-dihydroquinoline-3-carboxamido)aceticacid as an off-white solid.

Method 23:2-(4-Hydroxy-1-methyl-6-morpholino-2-oxo-1,2-dihydroquinoline-3-carboxamido)aceticacid

A solution of ethyl2-(6-bromo-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate(202 mg, 527 μmol), Pd₂dba₃.CHCl₃ (55 mg, 53 μmol), X-Phos (50 mg, 105μmol) and morpholine (115 μl, 1318 μmol) in 1,4-dioxane (3 ml) wastreated with sodium tert-butoxide (203 mg, 2109 μmol). The reaction wasstirred at 80° C. in a sealed tube. After 21 hours, the solution wasadsorped onto silica gel, concentrated in vacuo and purified by silicagel chromatography (eluant: 4% methanol/dichloromethane, followedby/dichloromethane+1% AcOH) and subsequently by RP HPLC (0-100%MeCN/water+1% TFA, 10 min) affording 21 mg (11%) of2-(4-hydroxy-1-methyl-6-morpholino-2-oxo-1,2-dihydroquinoline-3-carboxamido)aceticacid as an yellow solid.

Method 24:2-(4-Hydroxy-1-methyl-7-morpholino-2-oxo-1,2-dihydroquinoline-3-carboxamido)aceticacid

A solution of methyl2-(7-bromo-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate(100 mg, 271 μmol), Pd₂dba₃.CHCl₃ (28 mg, 27 μmol), X-Phos (26 mg, 54μmol) and morpholine (71 μl, 813 μmol) in 1,4-dioxane (3 ml) was treatedwith sodium tert-butoxide (104 mg, 1084 μmol). The reaction was stirredat 80° C. in a sealed tube. After 24 hours, the suspension was cooled to23° C., filtered through celite (extensively washing with methanol), thefiltrate concentrated in vacuo and purified by silica gel chromatographyafter adsorption onto silica (eluant: 10% methanol/dichloromethane+1%AcOH), affording 18 mg (18%) of2-(4-Hydroxy-1-methyl-7-morpholino-2-oxo-1,2-dihydroquinoline-3-carboxamido)aceticacid as a greenish-white solid.

Method 25:2-(4-Hydroxy-1-methyl-2-oxo-7-(piperidin-1-yl)-1,2-dihydroquinoline-3-carboxamido)aceticacid

A solution of tent-butyl2-(7-bromo-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate(205 mg, 498 μmol), Pd₂dba₃.CHCl₃ (52 mg, 50 μmol). X-Phos (48 mg, 100μmol) and piperidine (123 μl, 1246 μmol) in 1,4-dioxane (5 ml) wastreated with sodium tert-butoxide (192 mg, 1994 μmol). The reaction wasstirred at 80° C. in a sealed tube. After 15 hours, the solution wascooled to 23° C., adsorped onto silica gel, concentrated in vacuo andpurified by silica gel chromatography (eluant: 5%methanol/dichloromethane, followed by 5% methanol/dichloromethane+1%AcOH), affording an yellow solid which was 83% pure. The impure solidwas purified by RP HPLC (0-100% MeCN/water+1% TFA, 10 min), affording 83mg (46%) of the product as an yellow solid.

Method 26:Methyl-2-(4-hydroxy-1-methyl-2-oxo-7-(2-(trimethylsilyflethynyl)-1,2-dihydroquinoline-3-carboxamido)acetate

In a sealed tube was combined methyl2-(7-bromo-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate(0.75 g, 2.0 mmol), Dichlorobis(triphenylphosphine) palladium(II) (0.14g, 0.20 mmol), copper(I) iodide (0.077 g, 0.41 mmol),ethynyltrimethylsilane (1.4 ml, 10 mmol), andN-ethyl-N-isopropylpropan-2-amine (2.8 ml, 16 mmol) in tetrahydrofuran(20.0 ml, 2.0 mmol). The tube was flushed with Ar, sealed, and placed inan oil bath at 100° C. for 5 hours. The dark mixture was cooled to rt,filtered and washed with ethyl acetate (2×30 mL). The crude mixture wasconcentrated, adsorbed onto silica and purified by flash chromatography(15% to 40% EtOAc:Hex gradient) to afford the product as a solid (0.59g, 75% yield).

Method 27:2-(7-Cyano-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)aceticacid

In a sealed flask was combined methyl2-(7-bromo-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate(2.0 g, 5.4 mmol), 1,1′-bis(diphenylphosphino)ferrocene (0.48 g, 0.87mmol), copper cyanide (1.9 g, 22 mmol), Pd₂(dba)₃ (0.20 g, 0.22 mmol)and 1,4-dioxane (50.0 mL, 5.4 mmol). The flask was flushed with argon,and then tetraethylammonium cyanide (0.85 g, 5.4 mmol) was added. Aftersealing the tube and heating at 75° C. for 4 hours, the reaction wascooled to rt and then adsorbed onto silica. The crude reaction mixturewas purified using flash chromatography (15-70% EtOAc:Hex gradient) toafford the ester intermediate. The methyl ester was hydrolyzed by mixingthe solid with 5 N aqueous NaOH (5 mL) in THF (4 mL) for 4 hours. Themixture was acidified to pH 1 with 5 N HCl and the solid was collectedby filtration, washed with water (5×15 mL) and then with ether (2×5 mL).The solid was dried in a vacuum oven overnight at 50° C. to afford thedesired material (0.92 g, 56% yield).

Method 28:2-(7-Ethyl-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)aceticacid

To a stirred solution of methyl2-(4-hydroxy-1-methyl-2-oxo-7-(2-(trimethylsilyl)ethynyl)-1,2-dihydroquinoline-3-carboxamido)acetate(0.59 g, 1.5 mmol) in N,N-dimethylformamide (5.0 mL, 1.5 mmol) andmethanol (1.0 mL, 1.5 mmol) was added cesium fluoride (0.23 g, 1.5 mmol)under nitrogen. After stirring at room temperature for 1 hour, themixture was concentrated to remove solvents. The resulting solid wasadsorbed onto silica and purified using flash chromatography (15-80%EtOAc:Hex gradient) to afford a yellow solid. The solid was suspended inmethanol (10 mL) with Pd/C (20 mol %) and exposed to hydrogen from aballoon for 16 h. The crude reaction mixture was filtered throughCelite, and the filter pad was washed with dichloromethane (5'10 mL)under argon. The filtrate was concentrated to give a white solid thatwas further purified on silica by flash chromatography (100%chloroform). The solid was then treated with 5 N aqueous NaOH (3 mL) inTHF (3 mL) for 5 hours. The mixture was acidified to pH 1 using 5 Naqueous HCl, and the resulting precipitate was collected by filtration.After washing the solid with water (5×10 mL) and ether (2×10 ml), thedesired material was obtained after drying in a vacuum oven overnight at50° C. (0.21 g, 38% yield, 3 steps).

Method 29: Ethyl2-(6-cyano-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate

Ethyl2-(4-hydroxy-6-iodo-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate(0.200 g, 0.46 mmol), 1,1′-bis(diphenylphosphino)ferrocene (0.048 g,0.087 mmol), Copper cyanide (0.194 g, 2.2 mmol) andTris(dibenzylideneacetone)dipalladium (0.020 g, 0.022 mmol) in1,4-dioxane (2 ml) were combined in a 10 ml tube. Tetraethylammoniumcyanide (0.085 g, 0.54 mmol) and 1,4-dioxane (1 ml) were added and thetube was sealed and heated to 145° C. for 15 min under Argon in amicrowave (Personal Chemistry 300W). After cooling, the mixture wasfiltered and washed with methylene chloride (50 ml). The filtrate waswashed with deionized water (3×50 ml), then with brine (50 ml), driedover magnesium sulfate then concentrated and dried in vacuo. Flashcolumn chromatography (Silica gel, 0-100% methylene chloride in hexane)gave a solid which was washed with diethyl ether, filtered and dried invacuo to give ethyl2-(6-cyano-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate(0.135 g, 90% yield).

Method 30: Ethyl2-(6-(2,4-dimethylthiazol-5-yl)-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate

To a solution of ethyl2-(4-hydroxy-1-methyl-2-oxo-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2-dihydroquinoline-3-carboxamido)acetate(0.200 g, 0.465 mmol) in 1,4-dioxane/dimethylformamide (4:1, 5 ml) wasadded 5-bromo-2,4-dimethyl-1,3-thiazole (0.134 g, 0.697 mmol),tetrakis(triphenylphosphine)palladium(0) (0.0537 g, 0.0465 mmol) andsodium carbonate (0.349 ml, 0.697 mmol). The mixture was heated to 145°C. in a sealed tube under argon for 15 min in a microwave (PersonalChemistry 300W). By LC/MS the ratio of ethyl ester to acid to startingmaterial was 9:3:1. After cooling the mixture was diluted with deionizedwater (50 ml) and extracted with ethyl acetate (2×25 ml). The organicsolution was washed with deionized water (2×50 ml), then with brine (30ml), dried over Magnesium sulfate, concentrated and dried in vacuo togive 241 mg crude product. Flash column chromatography (silica gel,0-25% ethyl acetate in methylene chloride) yielded 86 mg of yellow solidwhich was washed with ether, filtered and dried in vacuo to give ethyl2-(6-(2,4-dimethylthiazol-5-yl)-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate(0.0330 g, 17.1% yield) as a white solid.

Method 31:2-(4-Hydroxy-1-methyl-2-oxo-6-phenyl-1,2-dihydroquinoline-3-carboxamido)aceticacid (can also be used for methyl ester)

To a solution of ethyl2-(4-hydroxy-1-methyl-2-oxo-6-phenyl-1,2-dihydroquinoline-3-carboxamido)acetate(120 mg, 315 μmol) and Tetrahydrofuran (15 ml) was added 5N Sodiumhydroxide (1.3 ml), and the mixture was stirred at room temperature.After 2.5 hours, reaction was complete. The reaction mixture wasacidfied with 5N HCL (2 ml) and concentrated on a roto-evaporator untilsolid appeared, then water was added and filtered to give the desiredcompound as a light peach colored solid. Yield=77 mg.

Method 32:2-(7-(3,5-dimethylisoxazol-4-yl)-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)aceticacid

Suspended Methyl2-(7-(3,5-dimethylisoxazol-4-yl)-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetatein 3 ml MeOH, 1 ml THF, and 2 ml 1M aqueous NaOH and stirred at 24° C.for 4 hours. The mixture was acidified to pH =1 using 2M aqueous HCl andthe solids collected by filtration, washed with H₂ O and dried in vacuo:50 mg white solids.

Method 33:2-(7-(4-(dimethylamino)phenyl)-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)aceticacid

Trifluoroacetic acid (4.00 ml, 54 mmol) was added to a suspension oftert-butyl2-(7-(4-(dimethylamino)phenyl)-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate(0.057g, 0.13 mmol) in dichloromethane (2.00 ml). After stirring at roomtemperature for 10 min water was added and the solution loaded in SCXMEGA BE column. The column was flushed with methanol extensible followedby 2M ammonia in methanol. The fractions obtained from the ammonia inmethanol were collected and the solvent removed in vacuo. The residuewas treated with 5N NaOH (2 ml) in THF (1 ml) and stirred at roomtemperature for 1 hour. The suspension was acifidied with 5N HCl and thesolids collected by filtration. The solids were washed with water,ether, dried in a vacuum oven at 50° C. to afford green solids (10 mg).

Method 34:2-(7-(3-cyanophenyl)-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)aceticacid

Trifluoroacetic acid (4.00 ml, 54 mmol) was added to a suspension oftert-butyl2-(7-(3-cyanophenyl)-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate(0.162 g, 0.37 mmol) in dichloromethane (2.00 ml). After stirring forone hour at room temperature water was added and the solids formed werecollected by filtration. The solids were washed with water, ether, driedin a vacuum oven at 50° C. to afford off-white solids in 74% yield.

Method 35:3-((carboxymethyl)carbamoyl)-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-7-carboxylicacid

Methyl3-((2-(benzyloxy)-2-oxoethyl)carbamoyl)-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-7-carboxylate(26 mg, 61 μmol) (65689-17-3) was dissolved in 12 ml dioxane/H₂O (5:1)and to this was added lithium hydroxide monohydrate (613 μl, 613 μmol)as a 1M aqueous solution. The resultant mixture was heated to 60 C for 4hrs. The solvent was removed in vacuo and the aqueous layer wasacidified with 2N HCl to pH2. Following dilution with EtOAc, the layerswere separated and the aqueous layer was extracted with EtOAc (3×). Theorganic layer was washed with H₂O and brine, then dried over Na₂SO₄. Thesolvent was removed by rotovap, azeotroping with benzene (3×) to give alight yellow solid which was rinsed with DCM followed by MeOH.

Method 36: 2-(1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)aceticacid

1-methyl-2-oxo-1,2-dihydroquinoline-3-carbonyl chloride, prepared from1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid (Archiv derPharmazie (1990), 323(2), 67-72) and oxalyl chloride, was added dropwiseto a solution of tert-butyl 2-aminoacetate hydrochloride (0.041 g, 0.25mmol), diisopropylethyl amine (0.086 ml, 0.49 mmol), in dichloromethane(1.00 ml), stirred at room temperature for 1 hr. The reaction mixturewas diluted with dichloromethane, washed with water and dried over MgSO4to afford tert-butyl2-(1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate in a 27%yield.

Trifluoroacetic acid (1.00 ml, 13 mmol) was added to tert-butyl2-(1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate (0.021 g,0.07 mmol) and stirred at room temperature for 15 minutes.Trifluoroacetic acid was removed under vacuum and the resulting solidswere washed with water (3×), ether (3×) and dried in a vacuum oven at50° C. to afford2-(1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetic acid in 29%yield.

Method 37:2-(4-Methoxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)aceticacid

Methyl2-(4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate(0.394 g, 1.4 mmol), methanol (0.33 ml, 8.1 mmol) and triphenylphosphine (0.94 ml, 4.1 mmol) were placed in a 50 mL round bottomedflask with 25 mL of THF. The flask was placed in an ice bath. Diethylazodicarboxylate (0.64 ml, 4.1 mmol) was added dropwise. A white solidwas filtered and this solid was purified by silica flash chromatography(0-3% MeOH/DCM) to give the desired product.

Methyl2-(4-methoxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetate(0.150 g, 0.5 mmol) was dissolved in THF in a 25 mL round bottom flask.NaOH was added and the mixture was stirred for 1.5 hours.Dichloromethane and water were added to the reaction and the layers wereseparated. The aqueous layer was washed two more times withdichloromethane. To the aqueous phase, IN HCL was added until the pH wasapproximately 1. The aqueous phase was then extracted with 25%IPA/CHCl3, dried with MgSO₄ and concentrated on a roto-evaporator. Thecompound was then purified by HPLC to give the desired product as awhite solid.

The following are examples of methods that may be used to quantitate HIFPHD activity and the inhibition of HIF PHD activity by compounds of thepresent invention.

Expression, Purification and Europium Labeling of VCB and Design of anEu-VCB based HTRF Assay for the Detection of Hydroxyprolyl HIF1αPeptides

The VCB complex is defined as the Von Hippel-Lindau protein (pVHL),elongin B and elongin C heterotrimeric complex. VCB specifically bindsto hydroxyproline residues of HIF1α, initiating polyubiquitinylation ofHIF1α and its subsequent proteolytic destruction. In the absence ofprolyl hydroxylase activity, VCB does not bind unmodified HIF1α. The VCBcomplex was expressed in E. coli and purified from the soluble fraction.The amino acid sequences of the three protein components are as follows:

VHL (Amino Acids 54-213) (SEQ ID NO: 1)MHHHHHHEAGRPRPVLRSVNSREPSQVIFCNRSPRVVLPVWLNFDGEPQPYPTLPPGTGRRIHSYRGHLWLFRDAGTHDGLLVNQTELFVPSLNVDGQPIFANITLPVYTLKERCLQVVRSLVKPENYRRLDIVRSLYEDLEDHPNVQKDLERLTQERIAHQRMGD ElonginB (SEQ ID NO: 2)MDVFLMIRRHKTTIFTDAKESSTVFELKRIVEGILKRPPDEQRLYKDDQLLDDGKTLGECGFTSQTARPQAPATVGLAFRADDTFEALCIEPFSSPPELPDVMKPQDSGSSANEQAVQ* ElonginC (Amino Acids 17-112) (SEQ ID NO: 3)MYVKLISSDGHEFIVKREHALTSGTIKAMLSGPGQFAENETNEVNFREIPSHVLSKVCMYFTYKVRYTNSSTEIPEFPIAPEIALELLMAANF LDCThe N-terminus of VHL contains a six histidine affinity tag forpurification purposes.

A VCB-based assay allows a highly sensitive and direct measurement ofenzymatic product formation (HIF1α protein or fragments thereofcontaining a hydroxylated proline residue) and is suitable for highthroughput screening.

For expression in E. coli, VHL 54-213 was cloned into pAMG21 (Pluxpromoter) between the NdeI-XhoI site. Immediately downstream of this isthe ElonginC gene cloned into the XhoI site to SacII. There is a 13 byspacer between the stop codon of VHL and the initiating codon ofElonginC. The expression plasmid pAMG21 is a 6118 base pair plasmid thatwas derived from the expression vector pCFM1656 (ATCC #69576), which inturn can be derived from the expression vector system described in U.S.Pat. No. 4,710,473. This design allows for chemical rather than thermalinduction of protein expression by substitution of the promoter region,replacing a synthetic bacteriophage lambda p1 promoter with a DNAsegment containing the LuxR gene and the LuxPR promoter, and affordsregulation of expression by the plasmid-encoded LuxR protein, therebyallowing any E. coli strain to serve as host.

ElonginB was cloned into pTA2 (pACYC 184.1 based vector) under thecontrol of a Lac promoter. Competent E. coli cells were transformed withthe pAMG21-VHL-ElonginC construct. These E. coli cells were renderedcompetent again prior to transformation with the pTA2-elonginB constructto produce the final E. coli strain containing both plasmid constructs.Induction of protein expression was initiated by the addition of IPTGand N-(3-oxo-hexanoyl)-homoserine lactone (HSL) at 30° C.

Bacterial cells were lysed by a microfluidizer in aqueous buffer of pH8.0 and the soluble fraction was separated by centrifugation. Thesoluble E. coli fraction was subjected to Nickel-NTA chelatingchromatography to utilize the six histidine affinity tag located on thepVHL construct. The pooled fractions from the nickel column were appliedto a Superdex 200 size exclusion chromatography (SEC) column. Theprotein eluted as a monomer on SEC, indicating that the three proteincomponents formed a complex in solution. The fractions from the SECcolumn were pooled and applied to a Q Sepharose anion exchange columnfor final purification. The purified complex was visualized by SDS-PAGEand the identities of the three protein components were confirmed byN-terminal amino acid sequencing.

Purified VCB was exchanged into 50 mM sodium carbonate buffer pH 9.2 andlabeled with a europium chelate overnight. LANCE™ europium chelate(PerkinElmer, Inc.; Eu-W1024 ITC chelate; catalog number is AD0013) wasused to label the lysine residues of the VCB complex. The chelatecontains an isothiocyanate reactive group that specifically labelsproteins on lysine residues (there are fifteen lysine residues in theVCB protein complex). The resulting europylated VCB was purified bydesalting columns and quantitated by standard means. The labeling yieldwas determined to be 6.6 europium groups per one VCB complex.

Two peptides were produced by SynPep, Inc.: a hydroxyproline modifiedpeptide and an unmodified control peptide. VCB was expected tospecifically bind to the hydroxyproline modified peptide (a mimic ofenzymatic hydroxylation by prolyl hydroxylase). VCB was not expected tobind to the unmodified peptide. Both peptides were produced with abiotin group at the N-terminus to allow for binding by thestreptavidin-labeled fluorescent acceptor allophycocyanin (streptavidinAPC; Prozyme, Inc.).

The sequence of the custom synthesized HIF1α peptides (amino acids556-575, with methionine residues replaced with alanine residues toprevent oxidation) were as follows:

(SEQ ID No: 4) (unmodified) Biotin-DLDLEALAPYIPADDDFQLR-CONH₂ (SEQ IDNo: 5) (modified) Biotin-DLDLEALA[hyP]YIPADDDFQLR-CONH₂

The peptides were purchased from SynPep as lyophilized solids and weresuspended in DMSO for experimental use. The peptides were quantitatedaccording to their absorbance at 280 nm.

Experiments were conducted in 96 well Costar polystyrene plates.Biotinylated peptides and europylated VCB were suspended in thefollowing buffer: 100 mM HEPES 7.5, 0.1 M NaCl, 0.1% BSA and 0.05% Tween20. The reagents were allowed to reach equilibrium by shaking for 1 hourbefore the plates were read on the Discovery Instrument (Packard). Thedata output is the ratio of the 665 nm and 620 nm emission signalresulting from the 320 nm excitation.

As shown in FIG. 1, the specific interaction of europylated VCB with thehydroxyproline modified HIF1αpeptide coupled to streptavidin APCgenerated a fluorescence signal detectable over the background signal.These results demonstrate a fluorescence signal generated by thespecific interaction of Eu-VCB with hyp-HIF1α peptide. Each barrepresents the data from a single well of a 96 well assay plate. Thesignal to background ratio was calculated from data from a control plate(unmodified peptide). Eu-VCB concentration was titrated across rows (nM)and streptavidin APC concentrations were titrated down columns. Thepeptide concentration was fixed at 100 nM.

Detection of Enzymatically Converted Hydroxyprolyl HIF-1α by HIF PHD2and Inhibition of HIF PHD2 Activity

Binding of the P564-HIF1α peptide to VCB was validated utilizing thehomogeneous time-resolved FRET (HTRF) technology. A 17 amino acid (17aa)peptide with an N-terminally labeled biotin molecule corresponding toamino acid sequences 558 to 574 of the HIF1α protein was synthesizedin-house (DLEMLAPYIPMDDDFQL) (SEQ ID No: 6). A second 17aa peptidecontaining a hydroxylated proline at position 564 was chemicallygenerated to mimic the PHD enzyme converted product form of the proteinthat is recognized by VCB. The assay was performed in a final volume of100 μl in buffer containing 50 mM Tris-HCl (pH 8), 100 mM NaCl, 0.05%heat inactivated FBS, 0.05% Tween-20, and 0.5% NaN₃. The optimal signalover background and the linear range of detection was determined bytitrating the hydroxylated or unhydroxylated peptide at variedconcentrations between 0 and 1 mM with a titration of VCB-Eu at varyingconcentrations between 0 and 50 nM with 50nM of streptavidin APC. Thebinding reagents were allowed to reach equilibrium by shaking for 1 hourbefore it was read on the Discovery Instrument (Packard). The dataoutput is the ratio of the 665 nm and 620 nm emission signal resultingfrom the 320 nm excitation.

HIF PHD2 activity was detected by P564-HIF1α peptide and VCB binding inthe HTRF format. HIF PHD2 was assayed at various concentrations between0 and 400 nM with 3 μM HIF1αpeptide in buffer containing 50 mM Tris-HCl(pH 7.5), 100 mM NaCl, 0.05% Tween 20, 2 mM 2-oxoglutarate (2-OG), 2 mMascorbic acid and 100 μM FeCl₂ in a final volume of 100 μL. Thetime-course was determined by periodically transferring 2.54 of thereaction into 2500 of 10× HTRF buffer containing 500 mM HEPES (pH 7.5),1M NaCl, 1% BSA, and 0.5% Tween-20 to terminate the enzyme reaction. 15nM HIF-1α peptide from the terminated reaction was added to 35 nMstreptavidin-APC and 10 nM VCB-Eu to a final volume of 100 μl in 10×HTRF buffer. The HTRF reagents were placed on a shaker for 1 hour beforedetection on the Discovery platform.

As demonstrated in FIG. 2, there was a dose dependent increase in HTRFsignal resulting from binding of the hydroxylated-P564-HIF1α peptide toVCB-Eu compared to the unhydroxylated form of the peptide resulting in a14 fold signal over noise ratio at 125 nM HIF1α peptide. VCB binding tothe APC bound peptide permits a FRET transfer between the Eu and APC.The signal was linear to 2 nM peptide with 3.125 nM VCB, but increasesto 62.5 nM peptide with 50 nM VCB resulting in a larger linear range.

HTRF detection utilizing Eu-labeled VCB is a practical system fordetermining HIF PHD2 catalytic activity. HIF PHD2 hydroxylation of theHIF1α peptide results in the increase affinity of VCB to the peptide andhence and increased FRET signal. As shown in FIG. 3, activity wasverified with a fairly linear and an increasing HTRF signal over time.There was a dose dependant increase in initial rates with increasing HIFPHD2 enzyme concentration up to 400 nM. The initial rates were linear to100 nM enzyme.

Inhibition of HIF PHD2 activity was quantified utilizing the HTRFtechnology. HIF PHD2 catalyzes a hydroxyl modification on the prolineresidue of the P564-HIF1α peptide substrate (Biotin-DLEMLAPYIPMDDDFQL)(SEQ ID No: 6) resulting in recognition and binding of the europylatedVon Hippel-Lindau protein (pVHL), elongin B and elongin C heterotrimeric(VCB-Eu) complex.

The PHD2 inhibition assay was executed by addition of freshly dissolvedFeCl₂ to 178.57 μM (100 μM final concentration) in PHD2 Reaction Buffercontaining 30 mM MES, pH 6, 10 mM NaCl, 0.25% Brij-35, 0.01% BSA, and 1%DMSO. 28 μL of the iron solution and 2 μl of inhibitor compoundsserially diluted in 100% DMSO (5% DMSO final) were added to blackpolypropylene 96-well microtiter plates. To that, 104 of 10 nM PHD2 (2nM final) was added to all wells of the plate except for the 8 wells ofcolumn 12 (LO control), and allowed to incubate at room temperature onthe shaker for one hour. Column 6 was the HI control containing PHD2enzyme and 5% DMSO vehicle, but no inhibitor compound. To initiate thePHD2 enzymatic reaction, 10 μL of a solution containing 500 nMP564-HIF1α peptide (100 nM final), 10 mM ascorbic acid (2 mM final), and1.25 μM 2-oxoglutarate (α-ketoglutarate; 0.25 μM final) in PHD2 ReactionBuffer was added to all wells of the plate and allowed to incubate onthe shaker at room temperature for one hour.

The reaction was terminated by addition of 25 μL HTRF Buffer (50 mMTRIS-HCl, pH 9, 100 mM NaCl, 0.05% BSA, and 0.5% Tween-20) containing150 mM succinate (product inhibitor; 50 mM final), 75 nMstreptavidin-APC (25 nM final), and 7.5 nM VCB-Eu (2.5 nM final). TheHTRF detection reagents were placed on a shaker for 1 hour to reachbinding equilibrium before reading on the Discovery platform(PerkinElmer). Europium is excited at 315 nm and phosphoresces at 615 nmwith a large Stoke's shift. APC, in turn, emits at 655 nm uponexcitation at 615 nm. The HTRF signal is measured as the ratio of theAPC 655 nm signal divided by the internal europium reference 615 nmemission signal.

The POC (percentage of control) was determined by comparing the signalfrom hydroxylated peptide substrate in the enzyme reaction containinginhibitor compound with that from PHD2 enzyme with DMSO vehicle alone(HI control), and no enzyme (LO control). POC was calculated using theformula: % control (POC)=(cpd−average LO)/(average HI−average LO)*100.Data (consisting of POC and inhibitor concentration in μM) was fitted toa 4-parameter equation (y=A+((B−A)/(1+((x/C)̂D))), where A is the minimumy (POC) value, B is the maximum y (POC), C is the x (cpd concentration)at the point of inflection and D is the slope factor) using aLevenburg-Marquardt non-linear regression algorithm.

In certain embodiments, compounds of the present invention exhibit a HIFPHD inhibitory activity IC₅₀ value of 40 μM or less. In additionalembodiments, compounds of the present invention exhibit a HIF PHDinhibitory activity IC₅₀ value of 10 μM or less.

Ruthenylation and Application of His-Tagged VCB inElectrochemiluminesence (ECL) Detection Assay

Ruthenylated VCB (Ru-VCB) was produced that retained HIF bindingactivity and was used to develop a bead-based electrochemiluminescenceassay for the detection of hydroxylated HIF peptides.

The following HIF1α peptides were synthesized (amino acids 558-574):

(SEQ ID No: 6) Biotin-HIF: DLEMLAPYIPMDDDFQL (SEQ ID No: 7)Biotin-HIF-OH: DLEMLA[hyP]YIPMDDDFQL

VCB, produced as described above, was ruthenylated (covalently throughlysine residues) by mixing 500 μL of VCB (1 mg/mL in 50 mM carbonatebuffer, pH 9.0) with 50 μL of ORI-TAG™-NHS ester (BioVeris Corporation,Gaithersburg, Md.; 3 mg/mL in 100% DMSO) for a 12:1 Ru:VCB molarchallenge ratio. The sample was wrapped in foil to protect it from lightand the chemical conjugation was allowed to occur for one hour at roomtemperature. The reaction was stopped by adding 20 μL 2M glycine andincubating for 10 minutes. Ru-VCB was purified from unconjugated Ru-tagby dialysis into storage buffer (20 mM Tris pH 7.5, 150 mM NaCl).

To evaluate the use of Ru-VCB as an ECL detection reagent forbiotin-HIF—OH (as well as to explore sensitivity and linear range), bothbiotin-HIF and biotin-HIF—OH were serially diluted and mixed withvarying concentrations of Ru-VCB and 0.33 ug/uL streptavidin M280Dynabeads (Invitrogen) in assay buffer (50 mM Tris-HCl, pH 8.0, 100 mMNaCl, 0.05% Tween 20, 0.5% NaN₃). After a two-hour incubation at roomtemperature with shaking, the reaction was read on the M-SERIES™analyzer (BioVeris Corporation, Gaithersburg, Md.). A low voltage wasapplied to the Ru-VCB/biotin-HIF—OH binding complexes, which in thepresence of Tripropylamine (TPA, the active component in the ECLreaction buffer, BV-GLOW™, BioVeris Corporation, Gaithersburg, Md.),resulted in a cyclical redox reaction generating light at 620 nm. Thesignal was detected on the Discovery platform.

FIG. 4 illustrates the Ru-VCB/biotin-HIF—OH binding curve and linearrange determination. Results are expressed as luminescence at 620 nm forRu-VCB plus biotin-HIF—OH divided by the signal from Ru-VCB plusbiotin-HIF. The assay can detect as little as 0.097 nM of hydroxylatedbiotin-HIF peptide standard (limit of detection=2×s/b) and is linear upto 1.56 nM.

Other embodiments of the present disclosure will be apparent to thoseskilled in the art from consideration of the specification and practiceof the present disclosure disclosed herein. It is intended that thespecification and examples be considered as exemplary only, with a truescope and spirit of the present disclosure being indicated by thefollowing claims.

TABLE 1 Cmpd Structure Name 1

Methyl 2-(4- hydroxy-1- methyl-2-oxo- 1,2-dihydroquinoline-3-carboxamido) acetate 2

2-(4-hydroxy-1- methyl-2-oxo-1,2- dihydroquinoline-3- carboxamido)aceticacid 3

2-(6-bromo-4-hydroxy- 1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 4

2-(6-chloro-4-hydroxy- 1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 5

(R)-2-(4-hydroxy-1- methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)propanoic acid 6

(S)-2-(4-hydroxy-1- methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)propanoic acid 7

Methyl 2-(4-hydroxy-6- iodo-1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetate 8

2-(4-hydroxy-6-iodo- 1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 9

2-(4-hydroxy-1-methyl- 2-oxo-6-phenyl-1,2- dihydroquinoline-3-carboxamido)acetic acid 10

2-(6-(4-tert-butylphenyl)- 4-hydroxy-1-methyl-2- oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 11

2-(4-hydroxy-1-methyl- 2-oxo-1,2- dihydroquinoline-3- carboxamido)-2-methylpropanoic acid 12

(R)-2-(4-hydroxy-1- methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)-3- methylbutanoic acid 13

methyl 2-(7-chloro-4- hydroxy-1-methyl-2- oxo-1,2- dihydroquinoline-3-carboxamido)acetate 14

1-(4-hydroxy-1-methyl-2- oxo-1,2- dihydroquinoline-3-carboxamido)cyclopropane- carboxylic acid 15

(S)-2-(4-hydroxy-1- methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)-3- methylbutanoic acid 16

2-(4-hydroxy-1- methyl-2-oxo-6-(4- (trifluoromethyl) phenyl)-1,2-dihydroquinoline-3- carboxamido)acetic acid 17

2-(4-hydroxy-1-methyl- 2-oxo-6-(3-(trifluoromethyl) phenyl)-1,2-dihydroquinoline-3- carboxamido)acetic acid 18

2-(6-(2-fluorophenyl)- 4-hydroxy-1-methyl-2- oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 19

2-(6-(3-fluorophenyl)- 4-hydroxy-1- methyl-2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 20

2-(6-(4-fluorophenyl)- 4-hydroxy-1-methyl- 2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 21

2-(4-hydroxy-6-(3- isopropylphenyl)-1- methyl-2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 22

2-(4-hydroxy-6-(4- methoxyphenyl)-1- methyl-2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 23

2-(4-hydroxy-1-methyl- 6-(naphthalen-2-yl)- 2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 24

2-(6-(benzo[b] thiophen-2-yl)-4- hydroxy-1-methyl-2- oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 25

2-(4-hydroxy-1-methyl- 2-oxo-6-(4-phenoxyphenyl)-1,2-dihydroquinoline-3- carboxamido)acetic acid 26

2-(6-(2-chlorophenyl)- 4-hydroxy-1-methyl- 2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 27

2-(6-(3-chlorophenyl)- 4-hydroxy-1-methyl-2- oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 28

2-(4-hydroxy-6- (1H-indol-5-yl)-1- methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 29

2-(7-chloro-4-hydroxy- 1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 30

methyl 2-(7-bromo-4- hydroxy-1-methyl- 2-oxo-1,2- dihydroquinoline-3-carboxamido)acetate 31

methyl 2-(4-hydroxy- 1-methyl-2-oxo-7- phenyl-1,2- dihydroquinoline-3-carboxamido)acetate 32

2-(7-bromo-4-hydroxy-1- methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 33

2-(4-hydroxy-1-methyl- 2-oxo-7-phenyl-1,2- dihydroquinoline-3-carboxamido)acetic acid 34

2-(4-hydroxy-6-(2- methoxyphenyl)-1- methyl-2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 35

2-(6-(4-chlorophenyl)- 4-hydroxy-1-methyl- 2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 36

2-(6-(3-formylphenyl)-4- hydroxy-1-methyl-2- oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 37

2-(4-hydroxy-6-(3- methoxyphenyl)-1- methyl-2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 38

(S)-3-hydroxy-2-(4- hydroxy-1-methyl-2- oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 39

2-(7-(3-chlorophenyl)- 4-hydroxy-1-methyl- 2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 40

2-(7-(4-chlorophenyl)- 4-hydroxy-1-methyl- 2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 41

N-(2-amino-2-oxoethyl)- 4-hydroxy-1-methyl- 2-oxo-1,2-dihydroquinoline-3- carboxamide 42

methyl 2-(4-hydroxy-1- methyl-2-oxo-7- (trifluoromethyl)-1,2-dihydroquinoline-3- carboxamido)acetate 43

(S)-methyl 2-(4-hydroxy- 1-methyl-2-oxo-7- (trifluoromethyl)-1,2-dihydroquinoline-3- carboxamido)propanoate 44

(S)-2-(4-hydroxy-1-methyl- 2-oxo-7- (trifluoromethyl)-1,2-dihydroquinoline-3- carboxamido)propanoic acid 45

(S)-2-(4-hydroxy-6-iodo- 1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)propanoic acid 46

2-(1-ethyl-4-hydroxy- 2-oxo-1,2- dihydroquinoline-3- carboxamido)aceticacid 47

2-(4-hydroxy-7-(4- methoxyphenyl)-1- methyl-2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 48

2-(4-hydroxy-1-methyl- 2-oxo-7- (trifluoromethyl)-1,2-dihydroquinoline-3- carboxamido)acetic acid 49

methyl 2-(4-hydroxy- 1,7-dimethyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetate 50

(S)-methyl 2-(4-hydroxy- 1,7-dimethyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)propanoate 51

(S)-2-(4-hydroxy-1,7- dimethyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)propanoic acid 52

2-(4-hydroxy-1,7- dimethyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 53

2-(4-hydroxy-1-methyl- 2-oxo-6-(2-phenyl)- phenyl-1,2-dihydroquinoline-3- carboxamido)acetic acid 54

(S)-2-(7-bromo-4- hydroxy-1-methyl- 2-oxo-1,2- dihydroquinoline-3-carboxamido)propanoic acid 55

(S)-methyl 2-(7-bromo- 4-hydroxy-1-methyl- 2-oxo-1,2-dihydroquinoline-3- carboxamido)propanoate 56

(S)-2-(4-hydroxy-1- methyl-2-oxo-6- phenyl-1,2- dihydroquinoline-3-carboxamido)propanoic acid 57

2-(4-hydroxy-1-methyl- 2-oxo-6- (pyridin-3-yl)-1,2- dihydroquinoline-3-carboxamido)acetic acid 58

2-(6-(2-chloro-5- methylpyrimidin-4- yl)-4-hydroxy-1- methyl-2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 59

(S)-methyl 2-(4-hydroxy- 8-methoxy-1- methyl-2-oxo-1,2-dihydroquinoline-3- carboxamido)propanoate 60

methyl 2-(4-hydroxy-8- methoxy-1-methyl-2- oxo-1,2- dihydroquinoline-3-carboxamido)acetate 61

2-(4-hydroxy-1-methyl- 2-oxo-6-(3-piperidin- 1-yl)phenyl)-1,2-dihydroquinoline-3- carboxamido)acetic acid 62

2-(4-hydroxy-1-methyl- 2-oxo-6-(3-(pyrrolidin- 1-yl)phenyl)-1,2-dihydroquinoline-3- carboxamido)acetic acid 63

ethyl 2-(4-hydroxy- 6-iodo-1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetate 64

(S)-2-(4-hydroxy- 8-methoxy-1- methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)propanoic acid 65

(S)-methyl 2-(5-fluoro- 4-hydroxy-1-methyl- 2-oxo-1,2-dihydroquinoline-3- carboxamido)propanoate 66

methyl 2-(5-fluoro- 4-hydroxy-1-methyl- 2-oxo-1,2- dihydroquinoline-3-carboxamido)acetate 67

2-(4-hydroxy-8-methoxy- 1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 68

methyl 2-(6-fluoro-4- hydroxy-1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetate 69

(S)-2-(5-fluoro-4- hydroxy-1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)propanoic acid 70

(S)-methyl 2-(6-fluoro-4- hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3- carboxamido)propanoate 71

2-(5-fluoro-4-hydroxy-1- methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 72

methyl 2-(4-hydroxy-1- methyl-2-oxo-5- (trifluoromethyl)-1,2-dihydroquinoline-3- carboxamido)acetate 73

(S)-methyl 2-(4-hydroxy-1- methyl-2-oxo-5- (trifluoromethyl)-1,2-dihydroquinoline-3- carboxamido)propanoate 74

(S)-2-(6-fluoro-4-hydroxy-1- methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)propanoic acid 75

methyl 2-(7-fluoro-4- hydroxy-1-methyl-2-oxo-1,2-1,2-dihydroquinoline-3- carboxamido)acetate 76

2-(4-hydroxy-1-methyl-2- oxo-5-(trifluoromethyl)-1,2-dihydroquinoline-3- carboxamido)acetic acid 77

2-(6-fluoro-4-hydroxy-1- methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 78

(S)-methyl 2-(7-fluoro-4- hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3- carboxamido)propanoate 79

(S)-2-(4-hydroxy-1-methyl-2- oxo-5-(trifluoromethyl)-1,2-dihydroquinoline-3- carboxamido)propanoic acid 80

(S)-2-(7-fluoro-4-hydroxy- 1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)propanoic acid 81

2-(6-(6-chloropyridin- 3-yl)-4-hydroxy-1- methyl-2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 82

2-(4-hydroxy-6-(3- hydroxyphenyl)-1- methyl-2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 83

2-(7-fluoro-4-hydroxy- 1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 84

2-(6-(2-cyclohexylphenyl)- 4-hydroxy-1-methyl- 2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 85

(S)-2-(4-hydroxy-1-methyl- 2-oxo-1,2- dihydroquinoline-3-carboxamido)butanoic acid 86

2-(4-hydroxy-1-methyl-2- oxo-6-(pyridin-4-yl)-1,2- dihydroquinoline-3-carboxamido)acetic acid 87

2-(4-hydroxy-1-methyl-2- oxo-6-(pyridin-2-yl)-1,2- dihydroquinoline-3-carboxamido)acetic acid 88

2-(4-hydroxy-1-methyl-2- oxo-7-o-tolyl-1,2- dihydroquinoline-3-carboxamido)acetic acid 89

methyl 3-((2-(benzyloxy)-2- oxoethyl)carbamoyl)-4-hydroxy-1-methyl-2-oxo-1,2- dihydroquinoline-7- carboxylate 90

3-((carboxymethyl) carbamoyl)-4-hydroxy-1- methyl-2-oxo-1,2-dihydroquinoline-7- carboxylic acid 91

2-(4-hydroxy-1-methyl-6- (naphthalen-1-yl)-2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 92

methyl 2-(4-hydroxy-1- methyl-2-oxo-7-(2- (trimethylsilyl)ethynyl)-1,2-dihydroquinoline-3- carboxamido)acetate 93

2-(4-hydroxy-1-methyl-2- oxo-7-p-tolyl-1,2- dihydroquinoline-3-carboxamido)acetic acid 94

2-(7-ethynyl-4-hydroxy-1- methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 95

2-(4-hydroxy-7-(2- methoxyphenyl)-1-methyl- 2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 96

2-(6-(2-formylphenyl)-4- hydroxy-1-methyl-1-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 97

2-(4-hydroxy-1-methyl-2- oxo-6-(quinolin-5-yl)-1,2- dihydroquinoline-3-carboxamido)acetic acid 98

2-(7-(2-chlorophenyl)-4- hydroxy-1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 99

(S)-2-(4-hydroxy-1,6- dimethyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)propanoic acid 100

ethyl 2-(4-hydroxy-1- methyl-6-(3-methylthiophen- 2-yl)-2-oxo-1,2-dihydroquinoline-3- carboxamido)acetate 101

methyl 2-(4-hydroxy-1- methyl-7-(3-methyl- thiophen-2-yl)-2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 102

ethyl 2-(4-hydroxy-1- methyl-2-oxo-6- (thiophen-2-yl)-1,2-dihydroquinoline-3- carboxamido)acetate 103

2-(4-hydroxy-1-methyl-6- (2-methylpyridin-3-yl)- 2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 104

2-(6-(3-chloro-5- (trifluoromethyl)pyridin- 2-yl)-4-hydroxy-1-methyl-2-oxo-1,2- dihydroquinoline-3- carboxamido)acetic acid 105

2-(4-hydroxy-1-methyl-7- (3-methylthiophen-2-yl)-2- oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 106

2-(4-hydroxy-1-methyl-2- oxo-7-(1H-pyrazol-4-yl)-1,2-dihydroquinoline-3- carboxamido)acetic acid 107

ethyl 2-(4-hydroxy-6-iodo- 1,7-dimethyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetate 108

2-(4-hydroxy-1,6-dimethyl- 2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 109

2-(4-hydroxy-1-methyl-6- (3-methylthiophen-2-yl)- 2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 110

(S)-methyl 2-(4-hydroxy-5- methoxy-1-methyl-2-oxo-1,2-dihydroquinoline-3- carboxamido)propanoate 111

methyl 2-(4-hydroxy-5- methoxy-1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetate 112

2-(4-hydroxy-6-iodo-1,7- dimethyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 113

(S)-methyl 2-(4-hydroxy-6- methoxy-1-methyl-2-oxo-1,2-dihydroquinoline-3- carboxamido)propanoate 114

methyl 2-(5-chloro-4- hydroxy-1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetate 115

ethyl 2-(4-hydroxy-1,7- dimethyl-2-oxo-6-phenyl-1,2- dihydroquinoline-3-carboxamido)acetate 116

2-(4-hydroxy-1-methyl- 2-oxo-7-(thiophen-3-yl)-1,2- dihydroquinoline-3-carboxamido)acetic acid 117

2-(4-hydroxy-7-(3- methoxyphenyl)-1- methyl-2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 118

2-(4-hydroxy-1-methyl-2- oxo-7-(thiophen-2-yl)-1,2- dihydroquinoline-3-carboxamido)acetic acid 119

2-(7-(3,5-dimethylisoxazol- 4-yl)-4-hydroxy-1-methyl- 2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 120

2-(4-hydroxy-1-methyl-2- oxo-6-(piperidin-1-yl)-1,2- dihydroquinoline-3-carboxamido)acetic acid 121

(S)-2-(4-hydroxy-5-methoxy- 1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)propanoic acid 122

2-(4-hydroxy-5-methoxy-1- methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 123

2-(4-hydroxy-1-methyl-2- oxo-6-(thiophen-2-yl)-1,2- dihydroquinoline-3-carboxamido)acetic acid 124

(S)-2-(4-hydroxy-6-methoxy- 1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)propanoic acid 125

ethyl 2-(6-(2,4-dimethyl- thiazol-5-yl)-4-hydroxy-1- methyl-2-oxo-1,2-dihydroquinoline-3- carboxamido)acetate 126

2-(5-chloro-4-hydroxy-1- methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 127

2-(4-hydroxy-1,7-dimethyl-2- oxo-6-phenyl-1,2- dihydroquinoline-3-carboxamido)acetic acid 128

2-(7-cyano-4-hydroxy-1- methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 129

2-(4-hydroxy-6-methoxy-1- methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 130

2-(6-(benzofuran-2-yl)-4- hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 131

2-(6-(2,4-dimethylthiazol-5- yl)-4-hydroxy-1-methyl-2- oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 132

methyl 2-(4-hydroxy-1,5- dimethyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 133

(S)-methyl 2-(4-hydroxy-1,5- dimethyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)propanoate 134

methyl 2-(7-(4-chloro- phenyl)-4-hydroxy-1- methyl-2-oxo-1,2-dihydroquinoline-3- carboxamido)acetate 135

(S)-2-(4-hydroxy-1,5- dimethyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)propanoic acid 136

2-(4-hydroxy-1,5- dimethyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 137

(S)-methyl 2-(5-chloro-4- hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3- carboxamido)propanoate 138

(S)-2-(4-hydroxy-1,8- dimethyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)propanoic acid 139

2-(4-hydroxy-1,8-dimethyl- 2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 140

(S)-2-(5-chloro-4-hydroxy-1- methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)propanoic acid 141

2-(7-(3-chloro-4-methoxy- phenyl)-4-hydroxy-1- methyl-2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 142

methyl 2-(5-bromo-4- hydroxy-1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 143

2-(4-hydroxy-1-methyl-2- oxo-7-(4-(trifluoro- methyl)phenyl)-1,2-dihydroquinoline-3- carboxamido)acetic acid 144

2-(4-hydroxy-1-methyl-2- oxo-7-(quinolin-3-yl)-1,2- dihydroquinoline-3-carboxamido)acetic acid 145

2-(4-hydroxy-1,7-dimethyl- 6-(2-methylpyridin-3-yl)- 2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 146

2-(5-bromo-4-hydroxy-1- methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 147

tert-butyl 2-(7-bromo-4- hydroxy-1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetate 148

2-(4-hydroxy-1-methyl-7- morpholino-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 149

2-(6-(2-bromophenyl)-4- hydroxy-1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 150

2-(7-ethyl-4-hydroxy-1- methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 151

2-(4-hydroxy-1-methyl- 2-oxo-6-(pyrimidin-5-yl)-1,2- dihydroquinoline-3-carboxamido)acetic acid 152

2-(6-(6-chloropyrimidin-4- yl)-4-hydroxy-1- methyl-2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 153

2-(4-hydroxy-1-methyl-2- oxo-6-(pyrimidin-2-yl)-1,2- dihydroquinoline-3-carboxamido)acetic acid 154

ethyl 2-(6-cyano-4-hydroxy- 1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetate 155

ethyl 2-(4-hydroxy-1-methyl- 6-(6-methylpyridazin-3-yl)- 2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 156

methyl 2-(4-hydroxy-1- methyl-2-oxo-7-p-tolyl-1,2- dihydroquinoline-3-carboxamido)acetate 157

2-(4-hydroxy-1-methyl-6- (6-methylpyridazin-3-yl)- 2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid 158

(S)-methyl 2-(4-hydroxy-1- methyl-7-nitro-2-oxo-1,2- dihydroquinoline-3-carboxamido)propanoate 159

(S)-2-(5-bromo-4-hydroxy-1- methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)propanoic acid 160

methyl 2-(4-hydroxy-1- methyl-7-nitro-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 161

tert-butyl 2-(4 hydroxy-6- iodo-1,7-dimethyl-2-oxo-1,2-dihydroquinoline-3- carboxamido)acetate 162

2-(4-hydroxy-1-methyl-2- oxo-7-(piperidin-1-yl)-1,2- dihydroquinoline-3-carboxamido)acetic acid 163

(S)-2-(4-hydroxy-1-methyl- 7-nitro-2-oxo-1,2- dihydroquinoline-3-carboxamido)propanoic acid 164

2-(6-cyano-4-hydroxy- 1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 165

2-(4-hydroxy-1-methyl-7- nitro-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 166

2-(4-hydroxy-1-methyl-6- morpholino-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 167

2-(7-(4-fluorophenyl)-4- hydroxy-1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 168

2-(7-(4-cyanophenyl)-4- hydroxy-1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 169

tert-butyl 2-(7-(4-(dimethyl- amino)phenyl)-4-hydroxy-1-methyl-2-oxo-1,2- dihydroquinoline-3- carboxamido)acetate 170

2-(7-(4-(dimethylamino) phenyl)-4-hydroxy-1-methyl- 2-oxo-1,2-dihydroquinoline-3- carboxamido)acetic acid hydrochloride 171

tert-butyl 2-(4 hydroxy-1,7- dimethyl-6-(3-methyl-thiophen-2-yl)-2-oxo-1,2- dihydroquinoline-3- carboxamido)acetate 172

2-(7-(3-cyanophenyl)-4- hydroxy-1-methyl-2-oxo-1,2- dihydroquinoline-3-carboxamido)acetic acid 173

2-(1-methyl-2-oxo-1,2- dihydroquinoline-3- carboxamido)acetic acid 174

2-(4-methoxy-2-oxo-1,2- dihydroquinoline-3- carboxamido)acetic acid 175

(S)-2-(4-hydroxy-1-methyl-2- oxo-7-(trifluoromethyl)-1,2-dihydroquinoline-3- carboxamido)propanoic acid Calc'd Cmpd M.W. (M + H)+1HNMR Method(s) 1 290.27 291 1H NMR (300 MHz, 7 (with tert- DMSO-d6) δppm 8.11 butyl ester); (1H, d, J = 7.7 Hz), 7.83 11, 15 (1H, t, J = 7.8Hz), 7.64 (1H, d, J = 8.2 Hz), 7.39 (1H, t, J = 7.67 Hz), 4.24 (2H, d, J= 5.7 Hz), 3.69 (3H, s), 3.65 (3H, s) 2 276.24 277 1H, NMR (300 MHz, 31DMSO-d6) δ ppm 10.57 (1H, t, J = 4.6 Hz), 8.09 (1H, d, J = 7.7 Hz), 7.82(1H, t, J = 7.3 Hz), 7.63 (1H, d, J = 8.5 Hz), 7.38 (1H, t, J = 7.5 Hz),4.14 (2H, d, J = 5.4 Hz), 3.64 (3H, s) 3 355.14 355 1H NMR (300 MHz, 4,8, 11, 15, DMSO-d6) δ ppm 10.50 31 (1H, t, J = 5.48 Hz), 8.14 (1H, d, J= 2.3 Hz), 7.96 (1H, dd, J = 9.1, 2.3 Hz), 7.61 (1H, d, J = 9.1 Hz),4.14 (2H, d, J = 5.6 Hz), 3.62 (3H, s) 4 310.69 311 1H NMR (300 MHz, 4,8, 11, 15, DMSO-d6) δ ppm 10.52 31 (1H, t, J = 4.8 Hz), 8.02 (1H, s),7.86 (1H, d, J = 9.1 Hz), 7.68 (1H, d, J = 8.5 Hz), 4.15 (2H, d, J = 5.4Hz), 3.63 (3H, s) 5 290.27 291 1H NMR (400 MHz, 15, 31 DMSO-d6): δ ppm10.75 (1H, d, J = 7.0 Hz), 6.07-8.16 (1H, m), 7.80-7.87 (1H, m), 7.65(1H, d, J = 8.6 Hz), 7.40 (1H, t, J = 7.6 Hz), 4.46-4.60 (1H, m, J =7.1, 7.1, 7.1 Hz), 3.65 (3H, s), 1.46 (3H, d, J = 7.2 Hz) 6 290.27 2911H NMR (400 MHz 15, 31 DMSO-d6): δ ppm 10.75 (1H, d, J = 7.0 Hz),8.07-8.16 (1H, m), 7.80-7.87 (1H, m), 7.65 (1H, d, J = 8.6 Hz), 7.40(1H, t, J = 7.6 Hz), 4.46-4.60 (1H, m, J = 7.1, 7.1, 7.1 Hz), 3.65 (3H,s), 1.46 (3H, d, J = 7.2 Hz) 7 416.17 417 NMR 300 (D6-DMSO): δ 15 ppm10.53 (1H, t, J = 3.0 Hz), 8.30 (1H, d, J = 2.0 Hz), 8.08 (1H, dd, J =3.0 Hz, 9.0 Hz), 7.45 (1H, d, J = 9.0 Hz), 4.23 (2H, d, J = 6.0 Hz),3.69 (3H, s), 3.60 (3H, s). 8 402.14 403 NMR 300 (D6-DMSO): δ 31 ppm12.98 (1H, br s), 10.50 (1H, br t), 8.31 (1H, d, J = 3.0 Hz), 8.08 (1H,dd, J = 3 Hz, J = 9.0 Hz), 7.45 (1H, d, J = 9 Hz), 4.13 (2H, d, J = 6.0Hz), 3.60 (3H, s). 9 352.34 353 NMR 300 (D6-DMSO): δ 19, 31 ppm 12.95(1H, br s), 10.58 (1H, br t), 8.31 (1H, br s), 8.15 (1H, m), 7.76 (3H,m), 7.52 (2H, m), 7.41 (1H, m), 4.15 (2H, d, J = 6 Hz), 3.69 (3H, s). 10408.45 409 NMR 300 (D6-DMSO): δ 19, 31 ppm 12.97 (1H, br s), 10.60 (1H,br m), 8.28 (1H, br s), 8.12 (1H, m), 7.70 (3H, m), 7.53 (2H, m), 4.16(2H, br m), 3.68 (3H, s), 1.33 (9H, s). 11 304.3 305 1H NMR (400 MHz,15, 31 DMSO-d6): δ ppm 12.82 (1H, s), 10.80-10.86 (1H, m), 8.10 (1H, d,J = 8.0 Hz), 7.83 (1H, t, J = 7.8 Hz), 7.65 (1H, d, J = 8.6 Hz), 7.39(1H, t, J = 7.5 Hz), 3.64 (3H, s), 1.57 (6H, s) 12 318.12 319 1H NMR(400 MHz, 15, 31 DMSO-d6): δ ppm 8.10 (1H, d, J = 9.4 Hz), 7.83 (1H, d),7.64 (1H, d), 7.40 (1H, dd), 0.95 (6H, d) 13 324.72 325 1H NMR (400 MHz,14 CHLOROFORM-d) δ ppm 10.65 (1H, s), 8.13 (1H, d, J = 8.4 Hz), 7.37(1H, s), 7.26 (1H, s), 4.24 (2H, d, J = 5.7 Hz), 3.80 (3H, s), 3.66 (3H,s). 14 302.28 303 15, 31 15 318.32 319 1H NMR (400 MHz, DMSO-d6): 15, 31δ ppm 10.84 (1H, d, J = 8.2 Hz), 8.10 (1H, dd, J = 8.0, 1.2 Hz),7.81-7.86 (1H, m), 7.66 (1H, d, J = 8.6 Hz), 7.40 (2H, t, J = 7.5 Hz),4.47 (1H, dd, J = 8.4, 4.5 Hz), 3.66 (3H, s), 0.97 (6H, dd, J = 6.8, 2.3Hz) 16 420.34 421 NMR 300 (D6-DMSO): δ ppm 19, 31 12.98 (1H, br s),10.58 (1H, br t), 8.40 (1H, d, J = 3.0 Hz), 8.25 (1H, dd, J = 3.0, J =9.0 Hz), 8.03 (2H, d, J = 9.0 Hz), 7.88 (2H, d, J = 9.0 Hz), 7.80 (1H,d, J = 9.0 Hz), 4.18 (2H, m), 3.72 (3H, s). 17 420.34 421 NMR 300(D6-DMSO): δ ppm 19, 31 10.63 (1H, br s), 8.42 (1H, s), 8.26 (1H, m),8.12 (2H, m), 7.88 (2H, d, J = 9.0 Hz), 7.80 (3H, m), 3.90 (2H, br s),3.73 (3H, s). 18 370.33 371 NMR 300 (D6-DMSO): δ ppm 19, 31 13.04 (1H,br s), 10.67 (1H, br t), 8.34 (1H, s), 8.13 (1H, d, J = 9.0 Hz), 7.87(1H, d, J = 9.0 Hz), 7.76 (1H, m), 7.57 (1H, m), 7.46 (2H, m), 4.26 (2H,m), 3.80 (3H, s). 19 370.33 371 NMR 300 (D6-DMSO): δ ppm 19, 31 13.02(1H, br s), 10.62 (1H, br t), 8.38 (1H, d, J = 3 Hz), 8.24 (1H, dd, J =3 Hz, J = 9.0 Hz), 7.80 (1H, d, J = 9.0 Hz), 7.71-7.56 (3H, m), 7.29(1H, m), 4.20 (2H, m), 3.74 (3H, s). 20 370.33 371 NMR 300 (D6-DMSO): δppm 19, 31 13.07 (1H, br s), 10.70 (1H, br t), 8.39 (1H, d, J = 3 Hz),8.25 (1H, dd, J = 3 Hz, J = 9.0 Hz), 7.96-7.91 (2H, m), 7.85 (1H, d, J =9.0 Hz), 7.46 (2H, m), 4.27 (2H, m), 3.81 (3H, s). 21 394.42 395 NMR 300(D6-DMSO): δppm 19, 31 12.80 (1H, br s), 10.44 (1H, br t), 8.14 (1H, d,J = 3 Hz), 8.00 (1H, dd, J = 3 Hz, J = 9.0 Hz), 7.58 (1H, d, J = 9.0Hz), 7.42 (2H, m), 7.29 (1H, t, J = 9.0 Hz), 7.15 (1H, d, J = 9.0 Hz),4.01 (2H, m), 3.54 (3H, s), 2.86 (1H, m), 1.13 (6H, d, J = 6.0 Hz). 22382.37 383 NMR 300 (D6-DMSO): δ ppm 19, 31 12.95 (1H, br s), 10.59 (1H,br t), 8.24 (1H, d, J = 3 Hz), 8.10 (1H, dd, J = 3 Hz, J = 9.0 Hz), 7.70(3H, m), 7.06 (2H, J = 9.0 Hz), 4.14 (2H, d, J = 6 Hz), 3.81 (3H, s),3.68 (3H, s). 23 402.4 403 NMR 300 (D6-DMSO): δ ppm 19, 31 12.96 (1H, brs), 10.59 (1H, br t), 8.46 (1H, d, J = 3 Hz), 8.34-8.29 (2H, m), 8.05(2H, d, J = 9.0 Hz), 7.96 (2H, m), 7.78 (1H, d, J = 9.0 Hz), 7.56 (2H,m), 4.16 (2H, m), 3.81 (3H, s). 24 408.43 409 NMR 300 (D6-DMSO): δ ppm19, 31 10.56 (1H, br s), 8.33 (1H, s), 8.21 (1H, m), 7.99 (2H, m), 7.87(1H, m), 7.71 (1H, m), 7.39 (2H, m), 3.86 (2H, br s), 3.66 (3H, s). 25444.44 445 NMR 300 (D6-DMSO): δ ppm 19, 31 12.93 (1H, br s), 10.58 (1H,br s), 8.27 (1H, d, J = 6.0 Hz), 8.13 (1H, dd, J = 3.0 Hz, J = 9.0 Hz),7.81-7.71 (3H, m), 7.44 (2H, m), 7.21-7.08 (5H, m), 4.15 (2H, m), 3.68(3H, s). 26 386.79 387 NMR 300 (D6-DMSO): δ ppm 19, 31 12.95 (1H, br s),10.57 (1H, br m), 8.11 (1H, br s), 7.92 (1H, m), 7.74 (1H, m), 7.62 (1H,m), 7.48 (3H, m), 4.15 (2H, m), 3.70 (3H, s). 27 386.79 387 NMR 300(D6-DMSO): δ ppm 19, 31 12.95 (1H, br s), 10.56 (1H, br m), 8.30 (1H, d,J = 3.0 Hz), 8.18 (1H, dd, J = 3.0 Hz, J = 9.0 Hz), 7.83 (1H, s), 7.73(2H, m), 7.56-7.46 (2H, m), 4.15 (2H, m), 3.68 (3H, s). 28 391.38 392NMR 300 (D6-DMSO): δ ppm 19, 31 12.95 (1H, br s), 11.21 (1H, br s),10.62 (1H, br t), 8.30 (1H, d, J = 3.0 Hz), 8.16 (1H, dd, J = 3.0 Hz, J= 9.0 Hz), 7.92 (1H, s), 7.71 (1H, d, J = 9.0 Hz), 7.51 (2H, m), 7.40(1H, m), 6.53 (1H, s), 4.16 (2H, m), 3.69 (3H, s). 29 310.69 311 1H NMR(400 MHz, DMSO-d6) δ ppm 31 10.42-10.53 (1H, m), 8.09 (1H, d, J = 8.6Hz), 7.73-7.76 (1H, m), 7.41- 7.46 (1H, m), 4.13 (2H, d, J = 5.5 Hz),3.60-3.66 (3H, m) 30 369.17 370 1H NMR (400 MHz, DMSO-d6) δ ppm 1410.43-10.57 (1H, m), 8.01 (1H, d, J = 8.4 Hz), 7.85-7.89 (1H, m), 7.54-7.58 (1H, m), 4.24 (2H, d, J = 5.9 Hz), 3.69 (3H, s), 3.63 (3H, s) 31366.37 367 1H NMR (400 MHz, DMSO-d6) δ ppm 21 10.57-10.62 (1H, m), 8.17(1H, d, J = 8.4 Hz), 7.88 (2H, d, J = 7.4 Hz), 7.79-7.82 (1H, m), 7.71(1H, d, J = 8.6 Hz), 7.53-7.59 (2H, m), 7.46-7.51 (1H, m), 4.25 (2H, d,J = 5.9 Hz), 3.74-3.78 (3H, m), 3.69-3.72 (3H, m) 32 355.14 355 1H NMR(400 MHz, DMSO-d6) δ ppm 31 10.39-10.51 (1H, m), 7.99 (1H, d, J = 8.6Hz), 7.82-7.88 (1H, m), 7.55 (1H, d, J = 8.6 Hz), 4.14 (2H, d, J = 5.5Hz), 3.59-3.67 (3H, m) 33 352.34 353 1H NMR (400 MHz, DMSO-d6) δ ppm 3110.57 (1H, t, J = 5.5 Hz), 8.17 (1H, d, J = 8.2 Hz), 7.88 (2H, d, J =7.4 Hz), 7.79-7.83 (1H, m), 7.70 (1H, dd, J = 8.4, 1.2 Hz), 7.53-7.59(2H, m), 7.46-7.51 (1H, m), 4.15 (2H, d, J = 5.7 Hz), 3.74-3.78 (3H, m)34 382.37 383 NMR 300 (D6-DMSO): δ ppm 19, 31 12.94 (1H, br s), 10.59(1H, br s), 8.16 (1H, s), 7.94 (1H, d, J = 9.0 Hz), 7.67 (1H, d, J = 9.0Hz), 7.40 (2H, m), 7.16 (1H, m), 7.07 (1H, m), 4.14 (2H, m), 3.80 (3H,s), 3.68 (3H, s). 35 386.79 387 NMR 300 (D6-DMSO): δ ppm 19, 31 12.94(1H, br s), 10.59 (1H, br s), 8.16 (1H, s), 7.94 (1H, d, J = 9.0 Hz),7.67 (1H, d, J = 9.0 Hz), 7.40 (2H, m), 7.16 (1H, m), 7.07 (1H, m), 4.14(2H, m), 3.68 (3H, s). 36 380.35 381 NMR 300 (D6-DMSO): δ ppm 19, 3112.96 (1H, br s), 10.56 (1H, br s), 10.14 (1H, s), 8.39 (1H, s), 8.30(1H, s), 8.23 (1H, m), 8.13 (1H, m), 7.93 (1H, m), 7.76 (2H, m), 4.15(2H, m), 3.69 (3H, s). 37 382.37 383 NMR 300 (D6-DMSO): δ ppm 19, 3110.63 (1H, br s), 10.14 (1H, s), 8.42 (1H, s), 7.76 (1H, d, J = 9.0 Hz),7.40- 7.19 (4H, m), 6.90 (1H, d, J = 9.0 Hz), 3.83 (3H, s), 3.52 (2H, brs), 3.50 (3H, s). 38 306.27 307 1H NMR (400 MHz, DMSO-d6): 15, 31 δ ppm10.84 (1H, d, J = 8.2 Hz), 8.10 (1H, dd, J = 8.0, 1.2 Hz), 7.81-7.86(1H, m), 7.66 (1H, d, J = 8.6 Hz), 7.40 (2H, t, J = 7.5 Hz), 4.47 (1H,dd, J = 8.4, 4.5 Hz), 3.92-3.76 (2H, m), 3.66 (3H, s). 39 386.79 387 1HNMR (400 MHz, DMSO-d6) δ ppm 21, 31 10.50-10.61 (1H, m), 8.17 (1H, d, J= 8.4 Hz), 7.96-8.02 (1H, m), 7.81- 7.89 (2H, m), 7.69-7.75 (1H, m),7.50- 7.63 (2H, m), 4.15 (2H, d, J = 5.5 Hz), 3.78 (3H, s) 40 386.79 3871H NMR (400 MHz, DMSO-d6) δ ppm 21, 31 10.51-10.60 (1H, m), 8.13-8.21(1H, m), 7.88-7.96 (2H, m), 7.78-7.84 (1H, m), 7.66-7.73 (1H, m),7.57-7.65 (2H, m), 4.15 (2H, d, J = 4.5 Hz), 3.70-3.81 (3H, m) 41 275.26276 1H NMR (300 MHz, DMSO-D6) 8, 11; 15 δ ppm 10.58 (1H, t, J = 4.9 Hz),8.10 (with (1H, d, J = 8.0 Hz), 7.82 (1H, t, glycinamide) J = 7.09 Hz),7.63 (1H, d, J = 8.3 Hz), 7.57 (1H, s), 7.38 (1H, t, J = 7.5 Hz), 7.21(1H, s), 4.02 (2H, d, J = 5.1 Hz), 3.64 (3H, s) 42 358.27 359 1H NMR(400 MHz, DMSO-d6) δ ppm 14 10.45-10.56 (1H, m), 8.28 (1H, d, J = 8.2Hz), 7.86-7.93 (1H, m), 7.69 (1H, d, J = 8.2 Hz), 4.20-4.31 (2H, m),3.67-3.72 (6H, m) 43 372.3 373 1H NMR (400 MHz, DMSO-d6) δ ppm 14 with10.61-10.70 (1H, m), 8.28 (1H, d, S-ala J = 8.4 Hz), 7.89-7.93 (1H, m),7.69 (1H, d, J = 8.4 Hz), 4.59-4.71 (1H, m), 3.67-3.73 (6H, m), 1.48(3H, d, J = 7.2 Hz) 44 358.27 359 1H NMR (400 MHz, DMSO-d6) δ ppm 3110.67 (1H, d, J = 6.8 Hz), 8.29 (1H, d, J = 8.2 Hz), 7.88-7.95 (1H, m),7.70 (1H, d, J = 8.4 Hz), 4.45-4.65 (1H, m), 3.64-3.75 (3H, m), 1.47(3H, d, J = 7.0 Hz) 45 416.17 417 1H NMR (300 MHz, DMSO-d6) δ ppm 14, 3113.12 (1H, s), 10.67 (1H, d, J = 7.0 Hz), 8.29 (1H, s), 8.07 (1H, dd, J= 8.9, 2.0 Hz), 7.45 (1H, d, J = 9.1 Hz), 4.45- 4.60 (1H, m), 3.59 (3H,s), 1.45 (3H, d, J = 7.2 Hz) 46 290.27 291 1H NMR (300 MHz, DMSO-d6) δppm 4 with Etl, 10.58 (1H, t, J = 5.48 Hz), 8.12 (1H, 7, 17, 31 dd, J =8.1, 1.4 Hz), 7.81 (1H, t, J = 7.2, 1.5 Hz), 7.68 (1H, d, J = 8.6 Hz),7.38 (1H, t, J = 7.4 Hz), 4.32 (2H, q, J = 7.0 Hz), 4.14 (2H, d, J = 5.6Hz), 1.23 (3H, t, J = 7.0 Hz) 47 382.37 383 1H NMR (400 MHz, DMSO-d6) δppm 21, 31 10.47-10.64 (1H, m), 8.10-8.18 (1H, m), 7.81-7.90 (2H, m),7.71-7.78 (1H, m), 7.63-7.71 (1H, m), 7.07-7.16 (2H, m), 4.09-4.18 (2H,m), 3.85 (3H, s), 3.75 (3H, s) 48 344.24 345 1H NMR (400 MHz, DMSO-d6) δppm 31 12.88-13.02 (1H, m), 10.44-10.54 (1H, m), 8.30 (1H, d, J = 8.2Hz), 7.88-7.93 (1H, m), 7.69 (1H, d, J = 8.4 Hz), 4.15 (2H, d, J = 5.7Hz), 3.67-3.74 (3H, m) 49 304.3 305 1H NMR (400 MHz, DMSO-d6) δ ppm 1410.54-10.63 (1H, m), 7.97 (1H, d, J = 8.2 Hz), 7.43-7.49 (1H, m), 7.21(1H, d, J = 8.2 Hz), 4.23 (2H, d, J = 5.9 Hz), 3.67-3.73 (3H, m), 3.60-3.65 (3H, m) 50 318.32 319 1H NMR (400 MHz, DMSO-d6) δ ppm 14 with S-10.64-10.78 (1H, m), 7.93 (1H, d, ala-OMe J = 8.2 Hz), 7.37-7.46 (1H,m), 7.18 (1H, d, J = 8.0 Hz), 4.55-4.68 (1H, m), 3.68-3.75 (3H, m),3.56-3.62 (3H, m), 2.45-2.51 (3H, m), 1.46 (3H, d, J = 7.0 Hz) 51 304.3305 1H NMR (400 MHz, DMSO-d6) δ ppm 31 10.72 (1H, d, J = 7.0 Hz), 7.99(1H, d, J = 8.2 Hz), 7.43-7.51 (1H, m), 7.18- 7.26 (1H, m), 4.42-4.59(1H, m), 3.58- 3.67 (3H, m), 1.44 (3H, d, J = 7.0 Hz) 52 290.27 291 1HNMR (400 MHz, DMSO-d6) δ ppm 31 10.46-10.63 (1H, m), 7.90-8.06 (1H, m),7.40-7.52 (1H, m), 7.17-7.29 (1H, m), 4.02 (2H, d, J = 5.1 Hz),3.60-3.66 (3H, m) 53 428.44 429 NMR 300 (D6-DMSO): δ ppm 19, 31 10.55(1H, br m), 7.85 (1H, s), 7.49 (3H, m), 7.45 (3H, m), 7.24 (3H, m), 7.15(2H, m), 3.88 (2H, m), 3.58 (3H, s) ppm. 54 369.17 370 1H NMR (400 MHz,DMSO-d6) δ ppm 31 10.63 (1H, d, J = 6.7 Hz), 7.98 (1H, d, J = 8.4 Hz),7.82-7.89 (1H, m), 7.51- 7.59 (1H, m), 4.45-4.59 (1H, m), 3.56- 3.68(3H, m), 1.45 (3H, d, J = 7.2 Hz) 55 383.19 384 1H NMR (400 MHz,DMSO-d6) δ ppm 14 with 10.62 (1H, d, J = 7.0 Hz), 7.96-8.03 S-ala (1H,m), 7.83-7.91 (1H, m), 7.50-7.60 (1H, m), 4.55-4.70 (1H, m), 3.67-3.73(3H, m), 3.59-3.65 (3H, m), 1.47 (3H, d, J = 7.2 Hz) 56 366.37 367 1HNMR (300 MHz, DMSO-d6) δ ppm 14 (with L- 13.10 (1H, s), 10.75 (1H, d, J= 7.2 alanine); Hz), 8.29 (1H, s), 8.15 (1H, dd, 19, 31 J = 9.0, 1.8Hz), 7.69-7.82 (3H, m), 7.52 (2H, t, J = 7.5 Hz), 7.41 (1H, t, J = 7.3Hz), 4.43-4.64 (1H, m), 3.68 (3H, s), 1.47 (3H, d, J = 7.0 Hz) 57 353.33354 1H NMR (300 MHz, DMSO-d6) δ ppm 19, 31 10.55 (1H, s), 9.17 (1H, s),8.75 (1H, d, J = 5.8 Hz), 8.49-8.61 (1H, m), 8.44 (1H, s), 8.27 (1H, d,J = 8.6 Hz), 7.81 (2H, d, J = 8.5 Hz), 4.16 (2H, d, J = 4.5 Hz), 3.70(3H, s) 58 402.79 403 NMR 300 (D6-DMSO): δ ppm 20, 31 10.55 (1H, br m),8.73 (1H, s), 8.44 (1H, s), 8.13 (1H, d, J = 9.0 Hz), 7.72 (1H, d, J =9.0 Hz), 3.82 (2H, m), 3.67 (3H, s), 2.43 (3H, s) ppm. 59 334.32 335 1HNMR (400 MHz, DMSO-d6): 20, 31 δ ppm 10.55 (1H, br m), 8.73 (1H, s),8.44 (1H, s), 8.13 (1H, d, J = 9.0 Hz), 7.72 (1H, d, J = 9.0 Hz), 3.82(2H, m), 3.67 (3H, s), 2.43 (3H, s) ppm. 60 320.3 321 1H NMR (400 MHz,DMSO-d6): 16 δ ppm 10.61 (1H, t, J = 5.7 Hz), 7.70 (1H, d, J = 7.8 Hz),7.45 (1H, d, J = 8.0 Hz), 7.32 (1H, t, J = 8.0 Hz), 4.23 (2H, d, J = 5.7Hz), 3.92 (3H, s), 3.81 (3H, s), 3.69 (3H, s) 61 435.47 436 1H NMR (300MHz, DMSO-d6) δ ppm 19, 31 10.54-10.61 (1H, m), 8.33 (1H, s), 8.16 (1H,d, J = 10.7 Hz), 7.74 (1H, d, J = 9.8 Hz), 7.36-7.59 (3H, m), 4.16 (2H,d, J = 5.0 Hz), 3.69 (3H, s), 3.35- 3.52 (4H, m), 1.71 (6H, d, J = 52.8Hz) 62 421.45 422 1H NMR (300 MHz, DMSO-d6) δ ppm 19, 31 10.59 (1H, t, J= 5.9 Hz), 8.26 (1H, s), 8.11 (1H, d, J = 10.5 Hz), 7.71 (1H, d, J = 8.5Hz), 7.29 (1H, t, J = 8.0 Hz), 6.95 (1H, d, J = 6.1 Hz), 6.82 (1H, s),6.61 (1H, d, J = 9.8 Hz), 4.16 (2H, d, J = 4.7 Hz), 3.69 (3H, s), 3.33(4H, t, J = 6.4 Hz), 1.99 (4H, t, J = 5.9 Hz) 63 430.19 431 NMR 300(D6-DMSO): 14 δ ppm 10.52 (1H, br m), 8.30 (1H, d, J = 3.0 Hz), 8.08(1H, dd, J = 3.0 Hz, J = 9.0 Hz), 7.45 (1H, d, J = 9.0 Hz), 4.22-4.12(4H, m), 3.61 (3H, s), 1.22 (3H, t, J = 6.0 Hz) ppm. 64 320.1 321 1H NMR(400 MHz, DMSO-d6): 31 δ ppm 10.76 (1H, d, J = 6.8 Hz), 7.70 (1H, d, J =8.0 Hz), 7.45 (1H, d, J = 8.0 Hz), 7.32 (1H, t, J = 8.0 Hz), 4.48-4.56(1H, m), 3.91 (3H, s), 3.81 (3H, s), 1.45 (3H, d, J = 7.0 Hz) 65 322.1323 1H NMR (400 MHz, DMSO-d6): 16 δ ppm 10.77 (1H, d, J = 6.8 Hz), 7.75-7.84 (1H, m), 7.46 (1H, d, J = 8.6 Hz), 7.16 (1H, dd, J = 11.6, 8.1 Hz),4.57- 4.67 (1H, m), 3.71 (3H, s), 3.63 (3H, s), 1.46 (3H, d, J = 7.2 Hz)66 308.08 309 1H, NMR (400 MHz, DMSO-d6): 16 δ ppm 10.62 (1H, t, J = 5.7Hz), 7.74- 7.84 (1H, m), 7.45 (1H, d, J = 8.6 Hz), 7.11-7.20 (1H, m),4.23 (2H, d, J = 5.9 Hz), 3.69 (3H, s), 3.63 (3H, s) 67 306.09 307 1HNMR (400 MHz, DMSO-d6): 31 δ ppm 10.58 (1H, t, J = 5.6 Hz), 7.70 (1H, d,J = 8.0 Hz), 7.44 (1H, d, J = 7.8 Hz), 7.32 (1H, t, J = 8.0 Hz), 4.13(2H, d, J = 5.5 Hz), 3.91 (3H, s), 3.81 (3H, s) 68 308.08 309 1H NMR(400 MHz, DMSO-d6): 16 δ ppm 10.56 10.66 (1H, m), 7.80 (1H, dd, J = 8.5,2.2 Hz), 7.69-7.75 (2H, m), 4.24 (2H, d), 3.78-3.82 (3H, m), 3.68-3.71(3H, m) 69 308.08 309 1H NMR (400 MHz, DMSO-d6): 31 δ ppm 10.76 (1H, d,J = 6.8 Hz), 7.74- 7.84 (1H, m), 7.45 (1H, d, J = 8.6 Hz), 7.15 (1H, dd,J = 11.8 , 8.1 Hz), 4.45- 4.56 (1H, m), 3.62 (3H, s), 1.45 (3H, d, J =7.2 Hz) 70 322.1 323 1H NMR (400 MHz, DMSO-d6): 16 δ ppm 10.68 10.79(1H, m), 7.76 (1H, d), 7.67-7.73 (1H, m), 4.57-4.69 (1H, m), 3.71 (3H,s), 3.62 (3H, s), 1.46 (3H, d, J = 7.2 Hz) 71 294.07 295 1H NMR (400MHz, DMSO-d6): 31 δ ppm 12.94 (1H, br. s.), 10.58-10.61 (1H, m),7.73-7.85 (1H, m), 7.46 (1H, d, J = 8.8 Hz), 7.11-7.21 (1H, m), 4.13(2H, d, J = 5.5 Hz), 3.62-3.65 (3H, s) 72 358.27 359 1H NMR (400 MHz,DMSO-d6): 16 δ ppm 10.67 10.78 (1H, m), 8.00-8.06 (1H, m), 7.92-7.99(1H, m), 7.82-7.87 (1H, m), 4.25 (2H, d, J = 5.3 Hz), 3.71 (3H, s), 3.70(3H, s) 73 372.3 373 1H NMR (400 MHz, DMSO-d6): 16 δ ppm 10.91 11.01(1H, m), 7.73-8.00 (2H, m), 7.34-7.40 (1H, m), 4.55-4.67 (1H, m), 3.70(3H, s), 3.66 (3H, s), 1.45 (3H, d, J = 7.0 Hz) 74 308.26 309 1H NMR(400 MHz, DMSO-d6): 31 δ ppm 10.75 (1H, d, J = 6.8 Hz), 7.79 (1H, d, J =8.8 Hz), 7.67-7.75 (2H, m), 4.46-4.59 (1H, m), 3.64 (3H, s), 1.46 (3H,d, J = 7.2 Hz) 75 308.26 309 1H NMR (400 MHz, DMSO-d6): 16 δ ppm 10.49(1H, t, J = 5.7 Hz), 8.15 (1H, dd, J = 8.8, 6.5 Hz), 7.54 (1H, dd, J =11.6, 2.2 Hz), 7.21-7.30 (1H, m), 4.22 (2H, d, J = 5.9 Hz), 3.69 (3H,s), 3.61 (3H, s) 76 344.24 345 1H NMR (400 MHz, DMSO-d6): 31 δ ppm 10.70(1H, t, J = 5.1 Hz), 7.99- 8.04 (1H, m), 7.95 (1H, t, J = 8.0 Hz), 7.84(1H, d, J = 7.4 Hz), 4.15 (2H, d, J = 5.7 Hz), 3.70 (3H, s) 77 294.24295 1H NMR (400 MHz, DMSO-d6): 31 δ ppm 10.57 (1H, t, J = 5.4 Hz), 7.80(1H, dd, J = 8.6, 2.5 Hz), 7.67-7.75 (2H, m), 4.14 (2H, d, J = 5.5 Hz),3.65 (3H, s) 78 322.29 323 1H NMR (400 MHz, DMSO-d6): 16 δ ppm 8.16 (1H,dd, J = 8.8, 6.7 Hz), 7.54 (1H, dd, J = 11.5, 2.0 Hz), 7.22- 7.30 (1H,m), 4.57-4.68 (1H, m), 3.70 (3H, s), 3.61 (3H, s), 1.38 (3H, d, J = 7.0Hz) 79 358.27 359 1H NMR (400 MHz, DMSO-d6): 31 δ ppm 10.89 (1H, d, J =7.0 Hz), 8.01- 8.06 (1H, m), 7.96 (1H, t, J = 8.1 Hz), 7.85 (1H, d, J =7.6 Hz), 4.48-4.60 (1H, m), 3.71 (2H, s), 3.17 (3H, s), 1.47 (3H, d, J =7.0 Hz) 80 308.26 309 1H NMR (400 MHz, DMSO-d6): 31 δ ppm 13.10 (1H, br.s.), 10.63 (1H, d, J = 6.8 Hz), 8.15 (1H, dd, J = 8.7, 6.6 Hz), 7.54(1H, dd, J = 11.5, 2.2 Hz), 7.25 (1H, td, J = 8.7, 2.1 Hz), 4.49- 4.56(1H, m, J = 7.2, 7.2, 7.2 Hz), 3.31 (3H, s), 1.45 (3H, d, J = 7.2 Hz) 81387.77 388 1H NMR (300 MHz, DMSO-d6) δ ppm 19, 31 10.55 (1H, t, J = 5.26Hz), 8.84 (1H, s), 8.37 (1H, s), 8.28 (1H, dd, J = 8.3 Hz), 8.21 (1H, d,J = 8.9 Hz), 7.77 (1H, d, J = 8.8 Hz), 7.63 (1H, d, J = 8.3 Hz), 4.15(2H, d, J = 5.4 Hz), 3.69 (3H, s). 82 368.34 369 1H NMR (300 MHz,DMSO-d6) δ ppm 19, 31 10.57 (1H, t, J = 5.5 Hz), 9.60 (1H, s), 8.24 (1H,d, J = 2.0 Hz), 8.08 (1H, dd, J = 8.8, 2.1 Hz), 7.71 (1H, d, J = 8.9Hz), 7.30 (1H, t, J = 7.9 Hz), 7.06-7.22 (2H, m), 6.80 (1H, d, J = 8.0Hz), 4.14 (2H, d, J = 5.6 Hz), 3.68 (3H, s). 83 294.24 295 1H NMR (400MHz, DMSO-d6): 31 δ ppm 12.92 (1H, br. s.), 10.46 (1H, t, J = 5.5 Hz),8.15 (1H, dd, J = 8.7, 6.4 Hz), 7.53 (1H, dd, J = 11.4, 2.1 Hz),7.22-7.28 (1H, m), 4.13 (2H, d, J = 5.7 Hz), 3.61 (3H, s) 84 434.48 43520, 31 85 304.3 305 1H NMR (400 MHz, DMSO-d6): 15, 31 δ ppm 8.23 (1H, d,J = 8.0 Hz), 7.96 (1H, t, J = 7.8 Hz), 7.77 (1H, d, J = 8.6 Hz), 7.52(1H, t, J = 7.5 Hz), 4.64 (1H, q, J = 6.5 Hz), 3.77 (3H, s), 1.88-2.10(2H, m), 1.06 (3H, t, J = 7.3 Hz) 86 353.33 354 NMR 300 (D6-DMSO): 15,31 δ ppm 10.49 (1H, br t), 8.89 (2H, d, J = 3.0 Hz), 8.62 (1H, s),8.45-8.39 (3H, m), 7.85 (1H, d, J = 9.0 Hz), 4.16 (2H, m), 3.71 (3H, s).87 353.33 354 NMR 300 (D6-DMSO): δ ppm 15, 31 10.53 (1H, br t), 8.82(1H, d, J = 3.0 Hz), 8.73 (1H, d, J = 9.0 Hz), 8.53 (1H, dd, J = 3.0 Hz,J = 9.0 Hz), 8.15 (1H, d, J = 9.0 Hz), 7.99 (1H, m), 7.77 (1H, d, J =9.0 Hz), 7.46 (1H, m), 4.16 (2H, m), 3.70 (3H, s). 88 366.12 367 NMR 400(D6-DMSO): δ ppm 21, 32 12.93 (1H, br. s), 10.58 (1H, br. t, J = 5.8Hz), 8.15 (1H, d, J = 8.2 Hz), 7.54 (1H, s), 7.37-7.34 (5H, m), 4.15(2H, d, J = 5.7 Hz), 3.68 (3H, s), 2.30 (3H, s). 89 424.4 [M − H] = 4231H NMR (300 MHz, CHLOROFORM- 13 d) δ ppm 10.75 (1H, t, J = 5.4 Hz), 8.27(1H, d, J = 8.5 Hz), 8.07 (1H, d, J = 1.1 Hz), 7.92 (1H, dd, J = 8.4,1.4 Hz), 7.31-7.41 (5H, m), 5.24 (2H, s), 4.28 (2H, d, J = 5.5 Hz), 4.00(3H, s), 3.75 (3H, s). 90 320.25 321 NMR 400 (D6-DMF): δ ppm 10.7 (1H,35 br. t, J = 5.3 Hz), 8.24 (1H, d, J = 8.3 Hz), 8.15 (1H, s), 7.94 (1H,d, J = 8.3 Hz), 4.31 (2H, d, J = 5.5 Hz), 3.77 (3H, s). 91 402.4 403 1HNMR (300 MHz, DMSO-d6) δ ppm 19, 31 10.68 (1H, s), 8.26 (1H, s), 8.01(1H, d, J = 7.6 Hz), 7.94 (1H, d, J = 8.3 Hz), 7.89 (1H, d, J = 8.5 Hz),7.37- 7.65 (6H, m), 3.52-3.58 (5H, m). 92 386.47 387 1H NMR (400 MHz,DMSO-d6) δ ppm 26 10.24-10.34 (1H, m), 7.81 (1H, d, J = 14.7 Hz),7.38-7.45 (1H, m), 7.15 (1H, d, J = 8.0 Hz), 3.99 (2H, d, J = 3.3 Hz),3.45 (3H, s), 3.41 (3H, s), 0.10 (9H, s) 93 366.37 367 1H NMR (400 MHz,DMSO-d6) δ ppm 31 12.81-13.00 (1H, m), 10.51-10.66 (1H, m), 8.10-8.22(1H, m), 7.73-7.85 (3H, m), 7.65-7.72 (1H, m), 7.30-7.41 (2H, m),4.06-4.24 (2H, m), 3.70-3.82 (3H, m), 2.32-2.44 (3H, m) 94 300.27 301 1HNMR (400 MHz, DMSO-d6) δ ppm 31 12.61-12.78 (1H, m), 10.16-10.33 (1H,m), 7.74-7.88 (1H, m), 7.41-7.53 (1H, m), 7.11-7.26 (1H, m), 4.35 (1H,m), 3.83-3.94 (2H, m), 3.11 (3H, s) 95 382.12 383 NMR 400 (D6-DMSO): δppm 12.92 21, 32 (1H, br. s), 10.58 (1H, br. m), 8.10 (1H, d, J = 8.4Hz), 7.65 (1H, s), 7.53- 7.47 (3H, m), 7.20 (1H, d, J = 8.2 Hz), 7.10(1H, t, J = 7.4 Hz), 4.14 (2H, d, J = 5.5 Hz), 3.81 (3H, s), 3.68 (3H,s). 96 380.35 381 1H NMR (300 MHz, DMSO-d6) δ ppm 19 10.42 (1H, t, J =4.1 Hz), 9.92 (1H, s), 8.12 (1H, d, J = 2.2 Hz), 7.91 (1H, d, J = 7.6Hz), 7.73-7.79 (1H, m), 7.51- 7.61 (3H, m), 7.37 (1H, d, J = 8.9 Hz),3.51 (3H, s), 3.48 (2H, d, J = 4.1 Hz) 97 403.39 404 1H NMR (300 MHz,DMSO-d6) δ ppm 19 10.58 (1H, t, J = 5.8 Hz), 8.97 (1H, dd, J = 4.1, 1.5Hz), 8.22 (1H, d, J = 8.9 Hz), 8.09-8.17 (2H, m), 7.95- 8.00 (1H, m),7.79-7.92 (2H, m), 7.66 (1H, d, J = 6.3 Hz), 7.56 (1H, dd, J = 8.6, 4.2Hz), 4.16 (2H, d, J = 5.7 Hz), 3.74 (3H, s) 98 386.07 387 NMR 400(D6-DMSO): δ ppm 21, 32 12.93 (1H, br. s), 10.56 (1H, br. t, J = 5.4Hz), 8.17 (1H, d, J = 8.2 Hz), 7.65-7.49 (6H, m), 4.15 (2H, d, J = 5.4Hz), 3.68 (3H, s). 99 304.11 305 1H NMR (400 MHz, DMSO-d6): 31 δ ppm10.78 (1H, d, J = 6.7 Hz), 7.89 (1H, s), 7.66 (1H, d), 7.55 (1H, d, J =8.6 Hz), 4.48-4.57 (1H, m), 3.62 (3H, s), 3.17 (3H, s), 1.45 (3H, d, J =7.0 Hz) 100 400.45 401 1H NMR (300 MHz, CDCl3) δ ppm 19 10.75 (1H, t, J= 5.3 Hz), 8.27 (1H, d, J = 2.1 Hz), 7.78 (1H, dd, J = 8.8, 2.2 Hz),7.39 (1H, d, J = 8.9 Hz), 7.24 (1H, d, J = 5.1 Hz), 6.95 (1H, d, J = 5.3Hz), 4.28 (2H, q, J = 7.0 Hz), 4.22 (2H, d, J = 5.5 Hz), 3.71 (3H, s),2.35 (3H, s), 1.31 (3H, t, J = 7.2 Hz) 101 386.09 387 NMR 400 (CDCl3): δppm 21 10.54 (1H, br. s), 8.23 (1H, d, J = 8.6 Hz), 7.43-7.41 (2H, m),7.32 (1H, d, J = 4.8 Hz), 6.99 (1H, d, J = 4.9 Hz), 4.25 (2H, d, J = 5.5Hz), 3.81 (3H, s), 3.72 (3H, s), 2.42 (3H, s). 102 386.42 387 1H NMR(300 MHz, CHLOROFORM- 19 d) δ ppm 10.75 (1H, t, J = 5.1 Hz), 8.39 (1H,d, J = 2.3 Hz), 7.91 (1H, dd, J = 8.7, 2.3 Hz), 7.36-7.42 (2H, m), 7.32(1H, dd, J = 5.2, 1.0 Hz), 7.11 (1H, d, J = 5.1, 3.8 Hz), 4.28 (2H, q, J= 7.2 Hz), 4.23 (2H, d, J = 5.5 Hz), 3.70 (3H, s), 1.32 (3H, t, J = 7.2Hz) 103 367.35 368 1H NMR (300 MHz, DMSO-d6) δ ppm 20, 31 10.51-10.63(1H, m), 8.62 (1H, d, J = 4.4 Hz), 8.09 (1H, s), 7.88-8.03 (2H, m), 7.77(1H, d, J = 8.9 Hz), 7.48-7.61 (1H, m), 4.15 (2H, d, J = 5.1 Hz), 3.70(3H, s), 2.54 (3H, s) 104 455.77 456 1H NMR (300 MHz, DMSO-d6) δ ppm 20,31 12.95 (1H, s), 10.38-10.64 (1H, m), 9.09 (1H, s), 8.63 (1H, d, J =1.3 Hz), 8.54 (1H, d, J = 2.0 Hz), 8.24 (1H, dd, J = 8.8, 2.1 Hz), 7.80(1H, d, J = 9.1 Hz), 4.16 (2H, d, J = 5.4 Hz), 3.70 (3H, s) 105 372.4373 NMR 400 (D6-DMSO): δ ppm 32 12.93 (1H, br. s), 10.54 (1H, br. t, J =5.3 Hz), 8.15 (1H, d, J = 8.2 Hz), 7.62 (1H, d, J = 5.2 Hz, 7.60 (1H,s), 7.49 (1H, br. d, J = 8.2 Hz), 7.10 (1H, d, J = 5.1 Hz), 4.13 (2H, d,J = 5.3 Hz), 3.69 (3H, s), 2.42 (3H, s). 106 342.31 343 NMR 400(D6-DMSO): δ ppm 21, 32 13.04 (2H, br. s), 10.55 (1H, br. t, J = 5.5Hz), 8.36 (2H, br. s), 8.03 (1H, d, J = 8.4 Hz), 7.77 (1H, s), 7.66 (1H,d, J = 8.4 Hz), 4.14 (2H, d, J = 5.5 Hz), 3.71 (3H, s). 107 444.22 4451H NMR (300 MHz, CHLOROFORM- 15 d) δ ppm 10.68 (1H, s), 8.58 (1H, s),7.22-7.27 (1H, m), 4.26 (2H, q, J = 7.2 Hz), 4.21 (2H, d, J = 5.5 Hz),3.65 (3H, s), 2.59 (3H, s), 1.31 (3H, t, J = 7.2 Hz) 108 290.09 291 1HNMR (400 MHz, DMSO-d6): 31 δ ppm 7.86-7.91 (1H, m), 7.61-7.68 (1H, m),7.50-7.56 (1H, m), 4.11 (2H, d, J = 5.9 Hz), 2.41 (3H, s) 109 372.4 3731H NMR (300 MHz, DMSO-d6) δ ppm 31 with 10.56 (1H, s), 8.09 (1H, s),7.90 (1H, LiOH d, J = 8.5 Hz), 7.72 (1H, d, J = 8.7 Hz), 7.52 (1H, d, J= 4.9 Hz), 7.05 (1H, d, J = 4.9 Hz), 4.15 (2H, d, J = 5.1 Hz), 3.67 (3H,s), 2.33 (3H, s) 110 334.12 335 1H NMR (400 MHz, DMSO-d6): 16 δ ppm10.87 (1H, d, J = 6.8 Hz), 7.71 (1H, t, J = 8.4 Hz), 7.15 (1H, d, J =8.6 Hz), 6.93 (1H, d, J = 8.0 Hz), 4.53- 4.63 (1H, m), 3.88 (3H, s),1.43 (3H, d, J = 7.2 Hz). 111 320.1 321 1H NMR (400 MHz, DMSO-d6): 16 δppm 10.73 (1H, t, J = 5.7 Hz), 7.71 (1H, t, J = 8.5 Hz), 7.15 (1H, d, J= 8.8 Hz), 6.93 (1H, d, J = 8.2 Hz), 4.19 (2H, d, J = 5.9 Hz), 3.88 (3H,s), 3.55 (3H, s) 112 416.17 417 1H NMR (400 MHz, DMSO-d6) δ ppm 31 10.50(1H, t, J = 4.0 Hz), 8.40 (1H, s), 7.61 (1H, s), 4.12 (2H, d, J = 4.0Hz), 3.62 (3H, s), 2.56 (3H, s) 113 334.12 335 1H NMR (400 MHz,DMSO-d6): 16 δ ppm 7.53-7.61 (2H, m), 7.45-7.49 (1H, m), 4.53-4.64 (1H,m), 3.84 (3H, s), 1.45 (3H, d, J = 7.0 Hz) 114 324.05 325 1H NMR (400MHz, DMSO-d6): 16 δ ppm 10.72 (1H, t, J = 4.7 Hz), 7.73 (1H, t, J = 8.1Hz), 7.62 (1H, d, J = 9.0 Hz), 7.43 (1H, d, J = 7.8 Hz), 4.24 (2H, d, J= 5.7 Hz), 3.70 (3H, s), 3.65 (3H, s) 115 394.42 395 1H NMR (400 MHz,CHLOROFORM- 19 d) δ ppm 10.78 (1H, s), 8.05 (1H, s), 7.33-7.46 (6H, m),4.26 (2H, q, J = 7.2 Hz), 4.22 (2H, d, J = 5.5 Hz), 3.72 (3H, s), 2.44(3H, s), 1.31 (3H, t, J = 7.1 Hz) 116 358.37 359 1H NMR (400 MHz,DMSO-d6) δ ppm 31 12.82-12.99 (1H, m), 10.48-10.61 (1H, m), 8.20-8.33(1H, m), 8.03-8.16 (1H, m), 7.69-7.90 (4H, m), 4.06-4.23 (2H, m), 3.75(3H, s) 117 382.37 383 1H NMR (400 MHz, MeOH) δ ppm 31 8.23 (1H, d, J =8.0 Hz), 7.68-7.72 (1H, m), 7.57-7.64 (1H, m), 7.38-7.45 (1H, m),7.31-7.36 (1H, m), 7.27-7.30 (1H, m), 6.97-7.05 (1H, m), 4.12-4.12 (2H,m), 3.89 (3H, s), 3.76 (3H, s) 118 358.37 359 1H NMR (400 MHz, DMSO-d6)δ ppm 31 12.87-13.02 (1H, m), 10.47-10.59 (1H, m), 8.05-8.14 (1H, m),7.84-7.91 (1H, m), 7.72-7.80 (2H, m), 7.63-7.69 (1H, m), 7.21-7.28 (1H,m), 4.14 (2H, d, J = 5.1 Hz), 3.70 (3H, s) 119 371.11 372 NMR 400(D6-DMSO): δ ppm 21, 32 12.93 (1H, s), 10.55 (1H, br. t, J = 5.0 Hz),8.17 (1H, d, J = 8.4 Hz), 7.58 (1H, s), 7.43 (1H, d, J = 8.4 Hz), 4.15(2H, d, J = 5.2 Hz), 3.68 (3H, s), 2.32 (3H, s). 120 359.38 360 1H NMR(400 MHz, DMSO-d6) δ ppm 22 10.67 (1H, br. s.), 7.61-7.71 (2H, m),7.46-7.60 (4H, m), 4.13 (2H, d, J = 5.5 Hz), 3.24 (4H, br. s.), 1.69(6H, br. s.) 121 320.1 321 1H NMR (400 MHz, DMSO-d6) δ ppm 31 10.88 (1H,d, J = 6.8 Hz), 7.71 (1H, t, J = 8.4 Hz), 7.16 (1H, d, J = 8.6 Hz), 6.94(1H, d, J = 8.2 Hz), 4.46-4.55 (1H, m), 3.90 (3H, s), 3.17 (3H, s), 1.44(3H, d, J = 7.2 Hz) 122 306.09 307 1H NMR (400 MHz, DMSO-d6): 31 δ ppm12.88 (1H, s), 10.71 (1H, s), 7.71 (1H, t, J = 8.0 Hz), 7.16 (1H, d, J =8.8 Hz), 6.94 (1H, d, J = 8.2 Hz), 4.11 (3H, d, J = 5.5 Hz), 3.90 (3H,s), 3.61 (3H, s) 123 358.37 359 NMR 1H NMR (300 MHz, DMSO-d₆) 31 with δppm 10.57 (1H, s), 8.23 (1H, s), 8.11 LiOH (1H, d, J = 3.0 Hz), 7.60(3H, m), 7.15-7.21 (1H, m), 4.15 (2H, d, J = 6.0 Hz), 3.66 (3H, s) 124320.1 321 1H NMR (400 MHz, DMSO-d6): 31 δ ppm 10.84 (1H, d, J = 6.7 Hz),7.61 (1H, d, J = 9.0 Hz), 7.42-7.51 (2H, m), 4.47-4.58 (1H, m), 3.86(3H, s), 3.63 (3H, s), 1.45 (3H, d, J = 7.2 Hz) 125 415.46 416 1H NMR(300 MHz, DMSO-d6) δ ppm 30 10.56 (1H, s), 8.05 (1H, s), 7.83 (2H, dd, J= 10.5, 8.3 Hz), 7.43 (1H, br. s.), 4.08-4.29 (4H, m), 3.67 (3H, s),2.64 (3H, s), 2.41 (3H, s), 1.25 (3H, t, J = 6.4 Hz) 126 310.04 311 1HNMR (400 MHz, DMSO-d6): δ ppm 10.70 (1H, t, J = 5.7 Hz), 7.72 (1H, t, J= 8.2 Hz), 7.62 (1H, d, J = 8.6 Hz), 7.42 (1H, d, J = 8.8 Hz), 4.14 (2H,d, J = 5.7 Hz), 3.64 (3H, s) 127 366.37 367 1H NMR (400 MHz, DMSO-d6) δppm 31 10.56 (1H, t, J = 5.5 Hz), 7.84 (1H, s), 7.58 (1H, s), 7.38-7.50(5H, m), 4.12 (2H, d, J = 5.5 Hz), 3.68 (3H, s), 2.42 (3H, s) 128 301.25302 1H NMR (400 MHz, DMSO-d6) δ ppm 27 12.87-13.05 (1H, m), 10.40-10.52(1H, m), 8.19-8.26 (2H, m), 7.73-7.79 (1H, m), 4.15 (2H, d, J = 5.3 Hz),3.67 (3H, s) 129 306.09 307 1H NMR (400 MHz, DMSO-d6): 31 δ ppm 10.67(1H, t, J = 5.4 Hz), 7.61 (1H, d, J = 9.2 Hz), 7.50 (1H, d, J = 2.5 Hz),7.43-7.48 (1H, m), 4.14 (2H, d, J = 5.5 Hz), 3.86 (3H, s), 3.64 (3H, s)130 392.36 393 1H NMR (300 MHz, DMSO-d6) δ ppm 19 10.53 (1H, s), 8.53(1H, s), 8.34 (1H, d, J = 6.3 Hz), 7.50-7.86 (4H, m), 7.18-7.42 (2H, m),3.98-4.18 (2H, m), 3.69 (3H, s) 131 387.41 388 1H NMR (300 MHz, DMSO-d6)δ ppm 31 10.54 (1H, s), 8.06 (1H, s), 7.88 (1H, d, J = 9.2 Hz), 7.74(1H, d, J = 8.0 Hz), 4.15 (3H, s), 3.06-3.25 (2H, m), 2.65 (3H, s),2.36-2.43 (3H, m) 132 304.11 305 1H NMR (400 MHz, DMSO-d6): 16 δ ppm10.77 (1H, t, J = 5.7 Hz), 7.65 (1H, t, J = 8.0 Hz), 7.48 (1H, d, J =8.8 Hz), 7.16 (1H, d, J = 7.2 Hz), 4.23 (2H, d, J = 5.7 Hz), 3.69 (3H,s), 3.63 (3H, s), 2.79 (3H, s) 133 318.12 319 1H NMR (400 MHz, DMSO-d6):16 δ ppm 10.94 (1H, d, J = 7.0 Hz), 8.03- 8.12 (1H, m), 7.66 (1H, t, J =7.4 Hz), 7.49 (1H, d, J = 8.8 Hz), 7.17 (1H, d, J = 7.4 Hz), 4.59-4.66(1H, m, J = 7.3, 7.3, 7.3 Hz), 3.67-3.73 (3H, m), 3.60- 3.65 (3H, m),2.76-2.81 (3H, m), 1.46 (1H, d, J = 7.2 Hz) 134 400.81 401 21 135 304.11305 1H NMR (400 MHz, DMSO-d6): 31 δ ppm 10.91 (1H, d, J = 7.0 Hz), 7.64(1H, t, J = 16.0 Hz), 7.47 (1H, d, J = 8.6 Hz), 7.15 (1H, d, J = 7.4Hz), 4.46-4.55 (1H, m), 3.62 (3H, s), 2.78 (3H, s), 1.45 (3H, d, J = 7.2Hz) 136 290.28 291 1H NMR (400 MHz, DMSO-d6): 31 δ ppm 7.65 (1H, t, J =8.6 Hz), 7.47 (1H, d, J = 8.4 Hz), 7.16 (1H, d, J = 7.4 Hz), 4.13 (2H,d, J = 5.5 Hz), 3.63 (3H, s), 1.91 (3H, s) 137 338.07 339 1H NMR (400MHz, DMSO-d6): 16 δ ppm 10.87 (1H, d, J = 6.7 Hz), 7.72 (1H, t, J = 8.1Hz), 7.60-7.64 (1H, m), 7.43 (1H, d, J = 7.6 Hz), 4.57-4.68 (1H, m, J =7.0, 7.0, 7.0 Hz), 3.70 (3H, s), 3.63 (3H, s), 1.46 (3H, d, J = 7.2 Hz)138 304.11 305 1H NMR (400 MHz, DMSO-d6): 31 δ ppm 10.68 (1H, d, J = 7.0Hz), 7.97 (1H, d, J = 8.4 Hz), 7.61 (1H, d, J = 7.2 Hz), 7.28 (1H, t, J= 7.6 Hz), 4.47-4.56 (1H, m), 3.72 (3H, s), 2.69 (3H, s), 1.45 (3H, d, J= 7.2 Hz) 139 290.09 291 1H NMR (400 MHz, DMSO-d6): 31 δ ppm 10.52 (1H,t, J = 5.6 Hz), 7.97 (1H, d, J = 7.6 Hz), 7.61 (1H, d, J = 7.8 Hz), 7.28(1H, t, J = 7.7 Hz), 4.13 (2H, d, J = 5.7 Hz), 3.72 (3H, s), 2.70 (3H,s) 140 324.05 325 1H NMR (400 MHz, DMSO-d6): 31 δ ppm 10.88 (1H, d, J =6.8 Hz), 7.72 (1H, t, J = 8.2 Hz), 7.62 (1H, d), 7.42 (1H, d, J = 7.6Hz), 4.49-4.57 (1H, m), 3.64 (3H, s), 1.46 (3H, d, J = 7.0 Hz) 141416.81 417 1H NMR (400 MHz, DMSO-d6) δ ppm 21, 31 12.93 (1H, s),10.48-10.61 (1H, m), 8.11 (1H, d, J = 8.2 Hz), 8.00 (1H, s), 7.84 (1H,d, J = 8.2 Hz), 7.75 (1H, s), 7.67 (1H, d, J = 8.2 Hz), 7.29 (1H, d, J =8.6 Hz), 4.14 (2H, d, J = 4.9 Hz), 3.94 (3H, s), 3.74 (3H, s) 142 368369/371 1H NMR (400 MHz, DMSO-d6): 16 δ ppm 8.16 (1H, d, J = 2.3 Hz),7.97 (2H, m), 7.62 (1H, d, J = 9 Hz,), 4.24 (2H, d, J = 5.7 Hz), 3.63(3H, s), 2.54 (3H, s) 143 420.34 421 1H NMR (400 MHz, DMSO-d6) δ ppm 21,31 10.22-10.77 (1H, m), 8.20 (1H, d, J = 8.6 Hz), 8.10 (2H, d, J = 7.6Hz), 7.83-7.94 (3H, m), 7.74 (1H, d, J = 8.6 Hz), 4.15 (2H, d, J = 5.7Hz), 3.76 (3H, s) 144 403.39 404 1H NMR (400 MHz, DMSO-d6) δ ppm 21, 3110.62 (1H, br s), 9.58 (1H, br s), 9.09 (1H, br s), 8.08-8.39 (4H, brm), 7.98 (2H, br s), 7.82 (1H, br s), 4.22 (2H, br s), 3.86 (3H, br s)145 381.38 382 1H NMR (400 MHz, DMSO-d6) δ ppm 19 10.42 (1H, br s), 8.40(1H, d, J = 4.0 Hz), 7.64 (1H, s), 7.53 (1H, s), 7.45 (1H, d, J = 8.0Hz), 7.20 (1H, dd, J = 8.0, 4.0 Hz), 3.98 (2H, br s), 3.56 (3H, s), 2.09(3H, s), 2.07 (3H, s) 146 353.99 355 1H NMR (400 MHz, DMSO-d6) δ ppm 15,31 10.50 (1H, s), 8.14 (1H, s), 7.95 (1H, dd, J = 9.0, 2.0 Hz), 7.60(1H, d, J = 9.2 Hz), 4.14 (2H, d, J = 5.5 Hz), 3.62 (3H, s). 147 410.1355.0, 357.0 NMR 400 (CDCl₃): δ ppm 10.64 15 (M − ^(t)Butyl + (1H, br.s), 8.06 (1H, d, J = 8.41 Hz) H)⁺ 7.54 (1H, d, J = 1.37 Hz), 7.42 (1H,dd, J = 8.61, 1.37 Hz) 4.12 (2H, d, J = 5.3 Hz) 3.66 (3H, s), 1.51 (9H,s) ppm. 148 361.35 362 1H NMR (400 MHz, DMSO-d6) δ ppm 24 10.47 (1H, br.s.), 7.88 (1H, d, J = 9.0 Hz), 7.05 (1H, d, J = 8.6 Hz), 6.74 (1H, br.s.), 4.08 (2H, d, J = 4.5 Hz), 3.76 (4H, br. s.), 3.56-3.61 (3H, m),3.42 (4H, br. s.) 149 431.24 433 1H NMR (300 MHz, DMSO-d6) δ ppm 19, 3112.95 (1H, s), 10.57 (1H, t, J = 5.5 Hz), 8.07 (1H, d, J = 2.0 Hz), 7.88(1H, dd, J = 8.8, 2.0 Hz), 7.79 (1H, d, J = 7.7 Hz), 7.73 (1H, d, J =8.8 Hz), 7.47-7.55 (2H, m), 7.33-7.41 (1H, m), 4.15 (2H, d, J = 5.6 Hz),3.69 (3H, s) 150 304.3 305 1H NMR (400 MHz, DMSO-d6) δ ppm 2810.50-10.60 (1H, m), 7.96-8.06 (1H, m), 7.44-7.49 (1H, m), 7.23-7.30(1H, m), 4.12 (2H, d, J = 5.3 Hz), 3.65 (3H, s), 2.80 (2H, q, J = 7.6,7.2 Hz), 1.27 (3H, t, J = 7.4 Hz) 151 354.31 355 1H NMR (300 MHz,DMSO-d6) δ ppm 20, 31 12.81 (1H, s), 10.40 (1H, t, J = 5.3 Hz),9.04-9.17 (3H, m), 8.30 (1H, s), 8.13 (1H, d, J = 8.3 Hz), 7.66 (1H, d,J = 8.6 Hz), 4.02 (2H, d, J = 5.4 Hz), 3.56 (3H, s) 152 388.76 389 1HNMR (300 MHz, DMSO-d6) δ ppm 20, 31 10.24-10.42 (1H, m), 8.99 (1H, s),8.84 (1H, s), 8.51 (1H, d, J = 8.5 Hz), 8.34 (1H, s), 7.65 (1H, d, J =8.9 Hz), 4.02 (2H, d, J = 5.4 Hz), 3.56 (3H, s) 153 354.32 355 1H NMR(300 MHz, DMSO-d6) δ ppm 20, 31 9.14 (1H, s), 8.94 (2H, d, J = 4.4 Hz),8.76 (1H, d, J = 8.8 Hz), 7.79 (1H, d, J = 9.5 Hz), 7.48 (1H, t, J = 4.3Hz), 4.15 (2H, d, J = 4.4 Hz), 3.69 (3H, s) 154 329.31 330 1H NMR (400MHz, DMSO-d6) δ ppm 29 10.41 (1H, s), 8.47 (1H, s), 8.19 (1H, d, J = 8.4Hz), 7.79 (1H, d, J = 8.8 Hz), 4.22 (2H, d, J = 4.9 Hz), 4.16 (2H, q, J= 6.9 Hz), 3.65 (3H, s), 1.22 (3H, t, J = 7.0 Hz) 155 396.4 397 1H NMR(400 MHz, DMSO-d6) δ ppm 30 10.51-10.61 (1H, m), 8.84 (1H, s), 8.57 (1H,d, J = 9.2 Hz), 8.27 (1H, d, J = 8.4 Hz), 7.81 (1H, d, J = 8.6 Hz), 7.68(1H, d, J = 9.0 Hz), 4.23 (2H, d, J = 4.3 Hz), 4.16 (2H, q, J = 7.4, 6.8Hz), 3.71 (3H, s), 2.68 (3H, s), 1.23 (3H, t, J = 6.9 Hz) 156 380.39 3811H NMR (400 MHz, DMF) δ ppm 21, 31 10.91-11.00 (1H, m), 8.36-8.42 (1H,m), 8.05-8.09 (1H, m), 7.98-8.04 (2H, m), 7.89-7.94 (1H, m), 7.53-7.61(2H, m), 4.54 (2H, d, J = 5.5 Hz), 4.04 (3H, s), 3.95 (3H, s), 2.61 (3H,s) 157 368.34 369 1H NMR (400 MHz, DMSO-d6) δ ppm 31 10.52 (1H, s), 8.83(1H, s), 8.56 (1H, d, J = 8.8 Hz), 8.40 (1H, d, J = 8.6 Hz), 7.83 (2H,d, J = 8.6 Hz), 4.15 (2H, d, J = 4.1 Hz), 3.70 (3H, s), 2.71 (3H, s) 158349.09 350 1H NMR (400 MHz, DMSO-d6): 16 δ ppm 8.29-8.35 (2H, m), 8.13(1H, dd, J = 8.9, 1.9 Hz), 4.59-4.68 (1H, m), 3.77 (3H, s), 3.70 (3H,s), 1.47 (3H, d, J = 7.2 Hz) 159 369.17 369 1H NMR (400 MHz, DMSO-d6):31 δ ppm 10.65 (1H, d, J = 7.2 Hz), 8.12- 8.16 (1H, m), 7.94 (1H, d, J =11.0 Hz), 7.59 (1H, d, J = 9.0 Hz), 4.46- 4.55 (1H, m), 1.44 (3H, d, J =7.0 Hz) 160 335.08 336 1H NMR (400 MHz, DMSO-d6): 16 δ ppm 10.50 (1H,s), 8.32-8.34 (1H, m), 8.30 (1H, s), 8.12 (1H, dd, J = 8.8, 2.0 Hz),4.25 (2H, d, J = 5.7 Hz), 3.72 (3H, s), 2.54 (3H, s) 161 472.27 417 1HNMR (400 MHz, CHLOROFORM- 15 (M − tBu) d) δ ppm 10.67 (1H, s), 8.56 (1H,s), 7.20-7.27 (1H, m, J = 18.2 Hz), 4.12 (2H, d, J = 5.3 Hz), 3.64 (3H,s), 2.58 (3H, s), 1.50 (9H, s) 162 359.38 360 1H NMR (400 MHz, DMSO-d6)δ ppm 25 10.48 (1H, t, J = 5.5 Hz), 7.84 (1H, d, J = 9.2 Hz), 7.01 (1H,dd, J = 9.2, 2.0 Hz), 6.68 (1H, d, J = 1.4 Hz), 4.10 (2H, d, J = 5.7Hz), 3.58 (3H, s), 3.49 (4H, br. s.), 1.63 (6H, br. s.) 163 335.08 3361H NMR (400 MHz, DMSO-d6): 31 δ ppm 13.17 (1H, br. s.), 10.62 (1H, d, J= 6.7 Hz), 8.34 (1H, s), 8.32 (1H, d, J = 9.0 Hz), 8.13 (1H, dd, J =8.7, 1.1 Hz), 4.50-4.59 (1H, m), 3.72 (3H, s), 1.47 (3H, d, J = 7.2 Hz)164 301.25 302 1H NMR (300 MHz, DMSO-d6) δ ppm 31 12.94 (1H, br. s.),10.40 (1H, s), 8.47 (1H, s), 8.17 (1H, d, J = 8.5 Hz), 7.81 (1H, d, J =8.9 Hz), 4.14 (2H, d, J = 4.3 Hz), 3.65 (3H, s) 165 321.06 322 1H NMR(400 MHz, DMSO-d6): 31 δ ppm 10.44 (1H, br. s.), 8.24-8.37 (2H, m), 8.12(1H, d, J = 8.4 Hz), 4.15 (2H, d, J = 5.3 Hz), 3.71 (3H, s) 166 361.35362 1H NMR (400 MHz, DMSO-d6) δ ppm 23 10.69 (1H, t, J = 5.3 Hz), 7.59(1H, dd, J = 2.5 Hz), 7.55 (1H, d, J = 9.2 Hz), 7.42 (1H, d, J = 2.3Hz), 4.13 (2H, d, J = 5.5 Hz), 3.78 (4H, t, J = 4.3 Hz), 3.62 (3H, s),3.13-3.19 (4H, m) 167 370.33 371 1H NMR (400 MHz, DMSO-d6) δ ppm 21, 3110.48-10.64 (1H, m), 8.16 (1H, d, J = 8.4 Hz), 7.88-7.99 (2H, m), 7.79(1H, s), 7.68 (1H, d, J = 8.2 Hz), 7.30- 7.44 (2H, m), 4.15 (2H, d, J =5.3 Hz), 3.75 (3H, s) 168 377.35 378 1H NMR (400 MHz, DMSO-d6) δ ppm 21,31 10.59 (1H, br s), 8.00-8.27 (5H, br m), 7.90 (1H, br s), 7.78 (1H, brs), 4.19 (2H, br s), 3.79 (3H, br s) 169 451.52 452 1H NMR (400 MHz,CHLOROFORM- 21 d) δ ppm 10.72-10.81 (1H, m), 8.19 (1H, d, J = 8.4 Hz),7.60 (2H, d, J = 8.6 Hz), 7.50 (1H, d, J = 8.6 Hz), 7.47 (1H, s), 6.82(2H, d, J = 8.4 Hz), 4.14 (2H, d, J = 5.1 Hz), 3.75 (3H, s), 3.04 (6H,s), 1.51 (9H, s) 170 395.41 396 1H NMR (400 MHz, DMSO-d6) δ ppm 21, 3310.54-10.66 (1H, br s), 8.12 (1H, br d, J = 9.4 Hz), 7.81 (2H, br d, J =8.2 Hz), 7.71-7.77 (1H, br s), 7.68 (1H, br d, J = 6.5 Hz), 6.89 (2H, brd, J = 6.8 Hz), 4.19 (2H, br d, J = 6.3 Hz), 3.78 (3H, br s), 3.04 (6H,br s) 171 442.53 443 1H NMR (400 MHz, DMSO-d6) δ ppm 19 10.53 (1H, brs), 7.85 (1H, s), 7.64 (1H, s), 7.55 (1H, d, J = 8.0 Hz), 7.05 (1H, d, J= 8.0 Hz), 4.11 (2H, d, J = 4.0 Hz), 3.67 (3H, s), 2.33 (3H, s), 2.01(3H, s), 1.45 (9H, s) 172 377.35 376 1H NMR (400 MHz, DMSO-d6) δ ppm 21,34 (M − H) 10.36-10.50 (1H, br s), 8.32 (1H, br s), 7.98-8.18 (2H, brm), 7.74-7.88 (2H, br m), 7.58-7.71 (2H, br d), 4.04 (2H, br s), 3.66(3H, br s) 173 260.24 261 1H NMR (400 MHz, DMSO-d6) 36 d ppm:12.26-13.26 (1H, br s), 9.75- 10.27 (1H, br s), 8.68-9.09 (1H, br s),7.95-8.22 (1H, br s), 7.49-7.91 (2H, br m), 7.11-7.46 (1H, br s),3.98-4.26 (2H, br s), 3.57-3.88 (3H, br s) 174 290.28 291 1H NMR (300MHz, DMSO-d6) d ppm 37 12.61 (1H, s), 8.77 (1H, t, J = 5.8 Hz), 7.95(1H, dd, J = 8.0, 1.3 Hz), 7.64- 7.72 (1H, m), 7.54 (1H, d, J = 8.2 Hz),7.27-7.34 (1H, m), 4.14 (3H, s), 3.94 (2H, d, J = 5.8 Hz), 3.58 (3H, s)185 358.08 359 1H NMR (400 MHz, CHLOROFORM- 15, 31 d): 10.71 (1H, d, J =6.7 Hz), 8.35 (1H, d, J = 8.4 Hz), 7.61 (1H, s), 7.55 (1H, d, J = 8.4Hz), 4.73-4.82 (1H, m, J = 7.0, 7.0, 7.0 Hz), 3.74 (3H, s), 1.63 (3H, d,J = 7.2 Hz)

1. A compound of the Formula I:

or a pharmaceutically acceptable salt thereof, wherein: n is 1 to 6; R₁is chosen from H, lower alkyl and substituted lower alkyl; R₂ is chosenfrom H, lower alkyl and substituted lower alkyl; R₃ and R₄ areindependently chosen from H, lower alkyl, substituted lower alkyl, lowerhaloalkyl, and substituted lower haloalkyl, wherein one of R₃ and R₄ isnot H; or R₃ and R₄ can join together to form a 3 to 6 membered ring ora substituted 3 to 6 membered ring; R₅ is chosen from OH, SH, NH₂, loweralkyl, substituted lower alkyl, lower alkoxy, substituted lower alkoxyand sulfanyl; R₆ is chosen from H, OH, SH, NH₂, NHSO₂R₁ and sulfonyl;and each of R₇, R₈, R₉ and R₁₀ is independently chosen from H, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, NR_(3′)R_(4′), C(O)OH, OR₁₃, SR₁₃,SO₂R₁₃, CN, NO₂, halo, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heteroarylalkyl, substituted heteroarylalkyl,heterocycloalkyl, substituted heterocycloalkyl, alkylsilyl, substitutedalkylsilyl, alkynylsilyl, substituted alkynylsilyl, alkoxy, substitutedalkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, and —X—R₁₂, wherein:R_(3′) and R_(4′) are independently chosen from H, lower alkyl,substituted lower alkyl, lower haloalkyl, and substituted lowerhaloalkyl; X is chosen from —N(R₁₁)—Y— and —Y—N(R₁₁)—; Y is chosen fromC(O), SO₂, alkylene, substituted alkylene, alkenylene, substitutedalkenylene, alkynylene, and substituted alkynylene; R₁₁ is chosen fromH, lower alkyl, and substituted lower alkyl; R₁₂ is chosen from H,heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl,heteroaryl and substituted heteroaryl; and R₁₃ is chosen from H, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl and NR_(3′)R_(4′); wherein optionally at least one of adjacentpairs R₆ and R₇, R₇ and R₈, R₈ and R₉, R₉ and R₁₀, and R₁₀ and R₁, jointogether to form a 4 to 7 membered ring or a substituted 4 to 7 memberedring.
 2. The compound according to claim 1, wherein R₃ and R₄ jointogether to form a 3 to 6 membered ring or a substituted 3 to 6 memberedring.
 3. The compound according to claim 2, wherein the 3 to 6 memberedring or the substituted 3 to 6 membered ring comprises at least oneheteroatom.
 4. The compound according to claim 2, wherein the 3 to 6membered ring or the substituted 3 to 6 membered ring comprises at leasttwo heteroatoms.
 5. The compound according to claim 1, wherein R₆ and R₇join together to form a 4 to 7 membered ring or a substituted 4 to 7membered ring.
 6. The compound according to claim 5, wherein the 4 to 7membered ring or the substituted 4 to 7 membered ring comprises at leastone heteroatom.
 7. The compound according to claim 5 wherein the 4 to 7membered ring or the substituted 4 to 7 membered ring comprises at leasttwo heteroatoms.
 8. The compound according to claim 5 wherein the 4 to 7membered ring or the substituted 4 to 7 membered ring comprises at leastthree heteroatoms.
 9. The compound according to claim 1, wherein atleast one of R₇, R₈, R₉ and R₁₀ is independently chosen from halo and amoiety substituted with at least one halo.
 10. The compound according toclaim 1, wherein at least one of R₇, R₈, R₉ and R₁₀ is independentlychosen from alkoxy or substituted alkoxy.
 11. The compound according toclaim 1, wherein at least one of R₇, R₈, R₉ and R₁₀ is independentlychosen from alkylsilyl, substituted alkylsilyl, alkynylsilyl, andsubstituted alkynylsilyl.
 12. The compound according to claim 1, whereinat least one of R₇, R₈, R₉ and R₁₀ is independently chosen from aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocycloalkyl,and substituted heterocycloalkyl.
 13. The compound according to claim 1,wherein at least one of R₇, R₈, R₉ and R₁₀ is independently chosen fromH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, andsubstituted alkynyl.
 14. The compound according to claim 1 having thestructure of any one of Compounds 5, 6, 11, 12, 14, 15, 38, 43-45, 50,51, 54-56, 59, 64, 65, 69, 70, 73, 74, 78, 79, 80, 85, 99, 110, 113,121, 124, 133, 135, 137, 138, 140, 158, 159, 163 and 175 set forth inTable
 1. 15. A pharmaceutical composition comprising at least onepharmaceutically acceptable excipient, and a therapeutically effectiveamount of a compound according to claim
 1. 16. The pharmaceuticalcomposition of claim 15, wherein the compound is present in an amounteffective for the treatment of at least one disease chosen fromischemia, anemia, wound healing, auto- transplantation, allo-transplantation, xeno-transplantation, systemic high blood pressure,thalassemia, diabetes, cancer and an inflammatory disorder.
 17. A methodof increasing HIF levels or activity in a subject comprisingadministering to the subject a compound according to claim
 1. 18-19.(canceled)
 20. A method of treating a hypoxic or ischemic relateddisorder in a subject comprising administering to a subject a compoundaccording to claim
 1. 21-23. (canceled)
 24. A method of treating adisease in a patient in need of such treatment comprising administeringto the patient a therapeutically effective amount of a compoundaccording to claim
 1. 25. The method according to claim 24, wherein thedisease is chosen from ischemia, anemia, wound healing, auto-transplantation, allo- transplantation, xeno-transplantation, systemichigh blood pressure, thalassemia, and diabetes. 26-33. (canceled)